Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Ultra-high-pressure liquid chromatography tandem mass spectrometry determination of hallucinogenic drugs in hair of psychedelic plants and mushrooms consumers Simona Pichini a,∗ , Emilia Marchei a , Oscar García-Algar b , Arelis Gomez b , Rita Di Giovannandrea a , Roberta Pacifici a a b
Drug Abuse and Doping Unit, Department of Therapeutic Research and Medicines Evaluation Istituto Superiore di Sanità, Roma 00161, Italy Unitat de Recerca Infància i Entorn (URIE), Paediatric Service, Institut de Recerca Hospital delMar – IMIM, Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 5 May 2014 Received in revised form 31 July 2014 Accepted 1 August 2014 Available online 10 August 2014 Keywords: Hair testing Mescaline N,N-dimethyltriptamine Psilocin Salvinorin A UHPLC–MS/MS
a b s t r a c t A procedure based on ultra-high-pressure liquid chromatography tandem mass spectrometry has been developed for the determination of mescaline, N,N-dimethyltryptamine, psilocin, psilocybin, salvinorin A in hair of consumers of psychedelic vegetal material such peyote or trichocereus cacti, psilocybe mushrooms, Salvia divinorum or psychedelic beverage ayahuasca. After hair washing with methyl alcohol and diethyl ether and subsequent addition of mescaline-d9 and 3,4-methylenedioxypropylamphetamine as internal standards, hair samples were treated with 250 l VMA-T M3 reagent for 1 h at 100 ◦ C. After cooling, 100 l M3 extract were diluted with 400 l water and a volume of 10 l was injected into chromatographic system. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a linear gradient elution with two solvents: 0.3% formic acid in acetonitrile and 5 mM ammonium formate pH 3. The mass spectrometer was operated in positive ion mode, using multiple reaction monitoring via positive electrospray ionization. The method was linear from the limit of quantification (0.03–0.05 ng/mg depending on analyte under investigation) to 10 ng/mg hair, with an intra- and inter-assay imprecision and inaccuracy always less than 15% and an analytical recovery between 79.6% and 97.4%, depending on the considered analyte. Using the validated method, mescaline was found in concentration range of 0.08–0.13 ng/mg in hair of peyote smokers, 3.2 ng salvinorin A per mg hair were determined in hair from a S. divinorum smoker, 5.6 ng N,N-dimethyltryptamine per mg hair from an ayahuasca user and finally 0.8 ng psilocybin per ng hair of a psilocybe consumer. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Even though currently “traditional drugs of abuse” (such as opioids, cocaine, cannabinoids and amphetamines) represents the 90% consumers’ demand, new psychotropic substances with stimulant or hallucinogenic properties have become increasingly popular among recreational drug users in recent years [1,2]. In particular, an increase in the consumption of vegetable substances with a hallucinogenic effects has been observed [3]. This group includes the well-known “magic” mushrooms of the species Psilocybe that contain psilocybin and psilocin; Peyote (Lophophora williamsii)
∗ Corresponding author at: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy. Tel.: +39 06 49906545; fax: +39 06 49902016. E-mail address: [email protected] (S. Pichini). http://dx.doi.org/10.1016/j.jpba.2014.08.006 0731-7085/© 2014 Elsevier B.V. All rights reserved.
and Trichocereus cacti rich in mescaline, Salvia divinorum leaves containing salvinorin A, as well as Mimosa hostilis roots bark or Ayahuasca decoction containing N,N-dimethyltryptamine [3–5]. These plants and mushrooms are obtainable not only in countries of origin [2], but also on internet web sites where, even if illegal in many countries, they can be easily bought and received anonymously avoiding normal law controls [6]. Buyers of psychedelic natural products look for mind-altering effects similar to those of LSD, acute perceptual changes (e.g. hearing colors and seeing sounds), subjective experiences, mystical experience with physiological effects similar to sympathetic arousal state [7]. Whereas analytical procedures for the determination of the above-reported hallucinogenic drugs are available in conventional biological matrices such as blood and urine [8–15], only mescaline has been successfully determined in a non conventional biological matrix such as hair to provide a reliable investigation tool to disclose a long-term abuse or past drug exposure [11].
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
In this concern, we developed and validated an ultra-highpressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to simultaneously identify and quantify mescaline, N,N-dimethyltryptamine, psilocin, psilocybin and salvinorin A in hair samples. The validated method was checked for its applicability in hair obtained from psychedelic plants and mushrooms consumers. 2. Experimental 2.1. Chemicals and materials Standard of mescaline (MES), N,N-dimethyltryptamine (DMT), psilocin (PSC), psilocybin (PSB), salvinorin A (SAL) and internal standards mescaline-d9 (MES-d9 ) and 3,4methylenedioxypropylamphetamine (MDPA) were obtained from Cellirant (Austin, TX, USA). VMA-T M3 (acidic aqueous buffer) reagent was provided by Comedical s.a.s. (Mattarello, Trento, Italy). Ultrapure water and all other reagents for UHPLC–MS/MS analytical grade were obtained from Sigma–Aldrich (Milan, Italy). 2.2. Hair samples Drug-free human hair samples obtained from 30 healthy individuals were analyzed during method validation to exclude any source of chromatographic interference and mixed to obtain a homogeneous pool of blank hair to be used for calibration standards and quality control (QC) samples. The entire length of hair shafts was collected from consumers of hallucinogenic plants and mushrooms. It consisted in 6 cm length each, from a female and male individuals smoking peyote cacti, a 2 cm hair shaft from a S. divinorum male smoker, a 2.5 cm hair shaft from an Ayahuasca male user and finally 1.5 cm hair shaft from a psilocybe male consumer were obtained by Pediatric Department of Hospital del Mar, Barcelona, Spain within the framework of a survey carried out with young consumers of hallucinogenic plants and mushrooms. 2.3. Calibration standards and quality control samples Stock standard solutions (1 mg/ml) were prepared in methyl alcohol and stored at −20 ◦ C. From stock solutions, working solutions of 10, 1 and 0.1 g/ml were prepared and used for the preparation of calibration curves and quality control samples. Working solutions of MES-d9 and MDPA at a concentration of 0.1 g/ml were also prepared in methyl alcohol and stored at −20 ◦ C. Calibration standards containing limit of quantification (LOQ) concentrations, 0.1, 0.5, 1.0, 5.0 and 10.0 ng analytes under investigation per mg hair were prepared daily for each analytical batch by adding suitable amounts of working solutions to 25 mg of prechecked drug-free hair pool.
285
Quality Control (QC) samples of 0.06, 4.0 and 8.5 ng analytes under investigation per mg hair were also daily prepared to be included in each analytical batch to check validation parameters (e.g. calibration, inaccuracy, imprecision, analytical recovery, etc.). 2.4. Sample preparation Aliquots of 25 mg finely cut hair samples were weighed in a glass test tube with hermetic cap. The samples were washed with two aliquots 5 ml methyl alcohol for five min and two aliquots 2.5 ml diethyl ether for other five minutes and then dried. They were then added with 10 l internal standards (MES d9 and MDPA 0.1 g/ml) and treated with 250 l M3 reagent for 1 h at 100 ◦ C in a thermoblock. Finally, the treated samples were cooled at room temperature and 100 l M3 extract were diluted with 400 l water and a volume of 10 l diluted extract was injected into the chromatographic system. 2.5. Ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) The analyses were carried out on an ultra-high pressure liquid chromatography system (Waters Acquity UHPLC, Waters Corporation, Milan, Italy) coupled with a triple quadrupole mass spectrometer (Waters Xevo TQ, Waters Corporation). Chromatographic separation was carried out on a Acquity UHPLC HSS C18 column (2.1 mm × 150 mm, 1.8 m) using a linear gradient elution with two solvents: 0.3% formic acid in acetonitrile (solvent A) and 5 mM ammonium formate pH 3 (solvent B). Solvent A was maintained at 5% for the first 0.50 min. It was increased to 55% from 0.50 to 10.00 min, then increased to 90% from 10.00 to 10.75 min, held at 90% from 10.75 to 12.85 min, and then decreased back to 5% from 12.85 to 13.00 min and held at 5% from 13.00 to 16.50 min for reequilibration. The flow rate was kept constant at 0.40 ml/min and the column temperature was set at 50 ◦ C. The separated analytes were identified and quantified with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 3 kV, desolvation temperature 600 ◦ C, source temperature 150 ◦ C, cone gas flow rate 20 l/h, desolvation gas flow rate 1000 l/h and collision gas flow rate 0.12 ml/min. Cone energy voltages, MRM transitions, and collision energy voltages were established for each analyte and the values are listed in Table 1. 2.6. Validation procedures Validation protocol applied in the present study included linearity, limits of detection (LOD) and quantification (LOQ), imprecision, inaccuracy, selectivity, carryover, matrix effect, recovery and process efficiency, as reported elsewhere [16,17]. Validation
Table 1 Ultra-performance liquid chromatography tandem mass spectrometry parameters for the multiple reaction monitoring (MRM) acquisition mode. Analytes
Retention time (min)
MRM transitions Quantification
Psilocybin Psilocin Mescaline N,N-dimethyltryptamine Salvinorin A Mescaline-d9 3,4-Methylenedioxypropylamphetamine
1.48 2.74 3.45 3.86 10.28 3.39 5.05
Confirmation
m/z
CV (V)
CE (eV)
m/z
CV (V)
CE (eV)
285.0 > 205.0 205.2 > 58.2 212.2 > 180.1 189.1 > 143.9 433.2 > 373.1 221.2 > 186.2 222.06 > 163.0
16 18 16 16 16 16 16
20 15 20 13 10 20 15
285.0 > 240.0 205.2 > 160.0 212.2 > 165.0 189.1 > 58.2 433.2 > 313.1 221.2 > 170.4 222.06 > 86.0
16 18 16 16 16 16 16
15 10 24 13 15 24 20
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289 1.48
Psilocybin m/z 285.0 > 205.0
% 0
2.74
100
Psilocin m/z 205.2 > 58.2
%
100
1.0
1.1
1.2
1.3
1.4
1.5
1.6
100
1.7
1.8
1.9
0
2.5
2.6
2.7
2.8
DMT m/z 189.1 > 143.9
% 0
Mescaline m/z 212.2 > 180.1
3.45
2.9
3.0
100
3.1
3.2
3.3
3.4
0 3.0
10.28
3.1
3.2
3.3
3.4 3.5
3.6
3.7
3.8
3.9
Salvinorin A m/z 433.2 > 373.1
%
3.86
100
%
286
3.5
3.6 3.7 3.8
3.9 4.0
4.1 4.2
4.3 4.4
0
10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9
Fig. 1. UHPLC–MS/MS chromatogram of 25 mg drug-free hair pool spiked with 0.1 ng analytes under investigation per mg hair.
parameters were calculated from five different daily replicates of quality control samples along five subsequent working days. Linearity was determined by least-squares regression with 1/2× weighting. Acceptable linearity was achieved when the coefficient of determination was at least 0.99 and the calibrators were quantified within ±20% at the LOQ and ±15% at other concentrations. The LOD and LOQ were evaluated with decreasing analyte concentrations in drug-spiked hair samples. The LOD was defined as the lowest concentration with acceptable chromatography, the presence of all transitions with signal-to-noise ratios of at least 3, and a retention time within ±0.2 min of the average retention time of the calibrator. LOQ was the lowest concentration that met LOD criteria and a signal-to-noise ratio of at least 10. Imprecision and inaccuracy were determined at the three QC samples concentrations by analyzing five replicates on three different days. Imprecision and inaccuracy, expressed as the coefficient of variation% of the measured values and error% respectively, were expected to be less than 20%. Selectivity tests were performed with obtained from 30 healthy individuals extracted and analyzed for assessment of potential interferences from endogenous substances. Furthermore, potential interferences from principal drugs of abuse such as opiates, cocaine and metabolites, cannabinoids, principal amphetamines and methylendioxyderivatives and ketamine, were also evaluated spiking 20 mg of blank hair with 200 ng of the aforementioned substances (corresponding to 10 ng/mg hair, the highest point of calibration curve) and carried through the entire procedure. The potential for carryover was investigated by injecting extracted blank hair pool sample, with added IS, immediately after
analysis of the highest concentration point of the calibration curve on each of the days of the validation protocol and measuring the area of eventual peaks, present at the retention times of analytes under investigation. Matrix effects, recovery and process efficiency were determined using the experimental design proposed by Matuszewski et al. [18]. Set 1 were five replicates of quality control material prepared in the mobile phase. Set 2 and 3 were five replicates of blank hair samples fortified with quality control material after and before extraction, respectively. Matrix effects were determined by dividing mean peak areas of set 2 by set 1 multiplied by 100. Recovery was determined by comparing the mean peak areas of analytes under investigation obtained in set 3 to those in set 2 multiplied by 100. Process efficiency expressed as the ratio of the mean peak area of an analyte spiked before extraction (set 3) to the mean peak area of the same analyte standards (set 1) multiplied by 100. 3. Results and discussion 3.1. Chromatography and validation parameters A representative ion chromatogram obtained following the extraction of 25 mg drug-free hair pool spiked with 0.1 ng analytes under investigation per mg hair is shown in Fig. 1. Chromatographic separation was set up using the mobile phase gradient program and flow rate already optimized for the determination of principal drugs of abuse in hair samples [17]. This was made in cooperation with instrument manufacturer (Waters) choosing the best chromatographic column and mobile phase which could, in the
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
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Table 2 Calibrations results, limits of detection (LOD) and limits of quantification (LOQ) for analytes under investigation. Analyte
Slopea
Psilocybin Psilocin Mescaline N,N-dimethyltryptamine Salvinorin A
7.400 3.536 1.140 7.970 2.807
a
Intercepta ± ± ± ± ±
−0.416 −0.527 −0.066 −0.034 −0.026
3.109 0.223 0.009 3.050 1.220
± ± ± ± ±
Correlation coefficient (r2 )a 0.342 0.114 0.048 0.210 0.100
0.998 0.995 0.999 0.997 0.997
± ± ± ± ±
0.002 0.004 0.001 0.003 0.002
LOD (ng/mg hair)
LOQ (ng/mg hair)
0.01 0.01 0.02 0.01 0.02
0.04 0.04 0.05 0.03 0.05
Mean ± standard deviation of four replicates.
Table 3 Intra-day (n = 5) and inter-day (n = 15) imprecision and inaccuracy for analytes under investigation. Intra-day imprecision (coefficient ofIntra-day inaccuracy (Error%) variation%) in quality control hair in quality control hair samples samples
Analyte
Inter-day imprecision (coefficient ofInter-day inaccuracy (Error%) variation%) in quality control hair in quality control hair samples samples
0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg Psilocybin Psilocin Mescaline N,Ndimethyltryptamine Salvinorin A
6.8 9.1 8.1 3.1
8.6 10.6 4.5 5.9
8.9 9.7 1.7 6.0
10.3 10.7 6.2 5.9
10.6 7.6 9.9 7.7
7.3 7.3 1.6 2.1
10.2 11.2 5.2 9.8
9.8 11.0 6.1 5.0
10.2 7.8 7.9 5.0
10.9 8.5 0.1 2.0
11.1 8.5 6.0 2.1
9.5 6.1 2.9 9.0
8.8
11.7
6.3
1.9
0.2
6.1
3.8
6.6
3.0
0.5
2.8
1.2
shortest possible run-time, separate and identify as many as possible psychoactive drugs and/or metabolites in different biological matrices. Linear calibration curves were obtained for the analytes under investigation with correlation coefficients (r2 ) always better than 0.99 in all cases and LODs and LOQs values were adequate for the purpose of the present study (Table 2). The intra- and inter-assay imprecision (measured as coefficient of variation, CV %) and inaccuracy (measured as percentage error) values were always lower than 12% (Table 3). No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. As reported in Table 4, matrix effect ranged from 79.3% to 99.2%, recovery from 79.6% to 97.4%, and process efficiency from 76.1% to 99.4%. 3.2. Analysis of hair samples In order to demonstrate its applicability on real samples, the method has been used to analyze hair samples obtained from obtained by Pediatric Department of Hospital del Mar, Barcelona, Spain within the framework of a survey carried out with young consumers of hallucinogenic plants and mushrooms. Fig. 2 shows the representative ion chromatograms of 5 different hair samples of users of these psychedelic vegetal material containing all the analytes under investigation.
Just in case of a female and a male peyote consumers, segmental hair analysis could be performed since hair strands were 6 cm in both cases. In female’s hair 0.08 and 0.11 mg mescaline per mg hair was found in the two first subsequent 2 cm segments, while the compound was absent in the third 2 cm segments. In case of male consumer, 0.12, 0.13 and 0.11 mg mescaline per mg hair were measured in the three 2 cm hair segments. In all the other cases, hair samples were obtained by male consumers with short hair (from 1.5 to 2.5 cm length) and segmental hair analysis could be not performed. In the single analyzed hair shaft, 3.2 ng salvinorin A per mg hair were found in the shaft from a S. divinorum smoker, 5.6 ng N,Ndimethyltriptamine per mg hair in the shaft from an Ayahuasca user and finally 0.8 ng psilocybin per ng hair in the shaft of a psilocybe consumer. No psilocin was found in this latter sample. Concerning segmental hair analysis of mescaline in the two samples coming from peyote consumers, past repeated consumption of cacti can confirmed, which in case of female dated in the last four months prior to analysis, in case of male extended to the last 6 months. Differently from our found concentrations varying between 0.08 and 0.13 ng mescaline per mg hair, Gambelunghe et al. reported a concentration of 1 ng/mg of mescaline in the first 2 cm shaft nearest to the scalp. The difference with our results can be hypothesized in the fact that in that case the subject consumed peyote tea while in our case the two consumers smoked dried cacti. No other information exist regarding the other hallucinogenic drugs included in our study. The analysis of other hair samples is a confirmation that the psychoactive principles N,Ndimethyltryptamine, psilocin, psilocybin, salvinorin A, can be found in the hair of Ayahuasca brew psilocybe mushrooms and
Table 4 Matrix effect, recovery and process efficiency data for analytes under investigation. Recovery (%) in quality control hair samples
Process efficiency (%) in quality control hair samples
Analyte
Matrix effect (%) in quality control hair samples 0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
Psilocybin Psilocin Mescaline DMT Salvinorin A
94.1 92.1 92.1 89.7 79.3
89.3 91.7 99.2 87.1 80.6
90.3 92.8 89.5 89.9 89.9
95.0 90.0 94.3 84.9 97.4
88.7 94.1 89.6 80.1 80.6
84.1 84.1 85.3 85.2 79.6
86.8 81.5 87.0 98.8 83.3
84.7 79.7 89.5 99.4 85.7
81.2 84.9 76.1 97.6 87.4
288
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
A 100
Psilocybin m/z 285.0 > 205.0
B 100
3.45
Mescaline m/z 212.2 > 180.1
%
%
1.48
3.85
0
0
1.6 1.7 1.8 1.9 DMT m/z 189.1 > 143.9
%
C 100
1.0 1.1 1.2 1.3 1.4
D
100
3.1
3.2
3.3
3.4
3.5
10.28
3.6
3.7
Salvinorin A m/z 433.2 > 373.1
%
0
3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4
0
10.1
10.3
10.5
10.7
10.0
Fig. 2. UHPLC–MS/MS chromatograms of 5 real hair samples of psychedelic plants and mushrooms consumers containing: (A) 0.8 ng psilocybin per mg hair; (B) 0.13 ng mescaline per mg hair; (C) 5.6 ng N,N-dimethyltryptamine per mg hair; (D) 3.2 ng salvinorin A per mg hair.
S. divinorum leaves consumers, respectively, evidencing a past consumption. No other conclusions can be drawn since these are preliminary results in a single short hair shafts. 4. Conclusion We have developed and validated an ultra-high-pressure liquid chromatography tandem mass spectrometry analysis of mescaline, N,N-dimethyltryptamine, psilocin, psilocybin, salvinorin A in hair of consumers of psychedelic vegetal material such peyote or trichocereus cacti, psilocybe mushrooms, S. divinorum or psychedelic beverage ayahuasca. applying a rapid digestion of the keratin matrix with M3 reagent. The method was tested for its feasibility in real samples and provided excellent results for determination of hallucinogenic drugs in hair of psychedelic plants and mushrooms consumers. Acknowledgements The authors thank Laura Martucci and Patrizia Gori from Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy for technical help. References [1] S.L. Hill, S.H. Thomas, Clinical toxicology of newer recreational drugs, Clin. Toxicol. (Phila) 49 (2011) 705–719, http://dx.doi.org/10.3109/ 15563650.2011.615318.
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Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Ultra-high-pressure liquid chromatography tandem mass spectrometry determination of hallucinogenic drugs in hair of psychedelic plants and mushrooms consumers Simona Pichini a,∗ , Emilia Marchei a , Oscar García-Algar b , Arelis Gomez b , Rita Di Giovannandrea a , Roberta Pacifici a a b
Drug Abuse and Doping Unit, Department of Therapeutic Research and Medicines Evaluation Istituto Superiore di Sanità, Roma 00161, Italy Unitat de Recerca Infància i Entorn (URIE), Paediatric Service, Institut de Recerca Hospital delMar – IMIM, Barcelona, Spain
a r t i c l e
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Article history: Received 5 May 2014 Received in revised form 31 July 2014 Accepted 1 August 2014 Available online 10 August 2014 Keywords: Hair testing Mescaline N,N-dimethyltriptamine Psilocin Salvinorin A UHPLC–MS/MS
a b s t r a c t A procedure based on ultra-high-pressure liquid chromatography tandem mass spectrometry has been developed for the determination of mescaline, N,N-dimethyltryptamine, psilocin, psilocybin, salvinorin A in hair of consumers of psychedelic vegetal material such peyote or trichocereus cacti, psilocybe mushrooms, Salvia divinorum or psychedelic beverage ayahuasca. After hair washing with methyl alcohol and diethyl ether and subsequent addition of mescaline-d9 and 3,4-methylenedioxypropylamphetamine as internal standards, hair samples were treated with 250 l VMA-T M3 reagent for 1 h at 100 ◦ C. After cooling, 100 l M3 extract were diluted with 400 l water and a volume of 10 l was injected into chromatographic system. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a linear gradient elution with two solvents: 0.3% formic acid in acetonitrile and 5 mM ammonium formate pH 3. The mass spectrometer was operated in positive ion mode, using multiple reaction monitoring via positive electrospray ionization. The method was linear from the limit of quantification (0.03–0.05 ng/mg depending on analyte under investigation) to 10 ng/mg hair, with an intra- and inter-assay imprecision and inaccuracy always less than 15% and an analytical recovery between 79.6% and 97.4%, depending on the considered analyte. Using the validated method, mescaline was found in concentration range of 0.08–0.13 ng/mg in hair of peyote smokers, 3.2 ng salvinorin A per mg hair were determined in hair from a S. divinorum smoker, 5.6 ng N,N-dimethyltryptamine per mg hair from an ayahuasca user and finally 0.8 ng psilocybin per ng hair of a psilocybe consumer. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Even though currently “traditional drugs of abuse” (such as opioids, cocaine, cannabinoids and amphetamines) represents the 90% consumers’ demand, new psychotropic substances with stimulant or hallucinogenic properties have become increasingly popular among recreational drug users in recent years [1,2]. In particular, an increase in the consumption of vegetable substances with a hallucinogenic effects has been observed [3]. This group includes the well-known “magic” mushrooms of the species Psilocybe that contain psilocybin and psilocin; Peyote (Lophophora williamsii)
∗ Corresponding author at: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy. Tel.: +39 06 49906545; fax: +39 06 49902016. E-mail address: [email protected] (S. Pichini). http://dx.doi.org/10.1016/j.jpba.2014.08.006 0731-7085/© 2014 Elsevier B.V. All rights reserved.
and Trichocereus cacti rich in mescaline, Salvia divinorum leaves containing salvinorin A, as well as Mimosa hostilis roots bark or Ayahuasca decoction containing N,N-dimethyltryptamine [3–5]. These plants and mushrooms are obtainable not only in countries of origin [2], but also on internet web sites where, even if illegal in many countries, they can be easily bought and received anonymously avoiding normal law controls [6]. Buyers of psychedelic natural products look for mind-altering effects similar to those of LSD, acute perceptual changes (e.g. hearing colors and seeing sounds), subjective experiences, mystical experience with physiological effects similar to sympathetic arousal state [7]. Whereas analytical procedures for the determination of the above-reported hallucinogenic drugs are available in conventional biological matrices such as blood and urine [8–15], only mescaline has been successfully determined in a non conventional biological matrix such as hair to provide a reliable investigation tool to disclose a long-term abuse or past drug exposure [11].
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
In this concern, we developed and validated an ultra-highpressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to simultaneously identify and quantify mescaline, N,N-dimethyltryptamine, psilocin, psilocybin and salvinorin A in hair samples. The validated method was checked for its applicability in hair obtained from psychedelic plants and mushrooms consumers. 2. Experimental 2.1. Chemicals and materials Standard of mescaline (MES), N,N-dimethyltryptamine (DMT), psilocin (PSC), psilocybin (PSB), salvinorin A (SAL) and internal standards mescaline-d9 (MES-d9 ) and 3,4methylenedioxypropylamphetamine (MDPA) were obtained from Cellirant (Austin, TX, USA). VMA-T M3 (acidic aqueous buffer) reagent was provided by Comedical s.a.s. (Mattarello, Trento, Italy). Ultrapure water and all other reagents for UHPLC–MS/MS analytical grade were obtained from Sigma–Aldrich (Milan, Italy). 2.2. Hair samples Drug-free human hair samples obtained from 30 healthy individuals were analyzed during method validation to exclude any source of chromatographic interference and mixed to obtain a homogeneous pool of blank hair to be used for calibration standards and quality control (QC) samples. The entire length of hair shafts was collected from consumers of hallucinogenic plants and mushrooms. It consisted in 6 cm length each, from a female and male individuals smoking peyote cacti, a 2 cm hair shaft from a S. divinorum male smoker, a 2.5 cm hair shaft from an Ayahuasca male user and finally 1.5 cm hair shaft from a psilocybe male consumer were obtained by Pediatric Department of Hospital del Mar, Barcelona, Spain within the framework of a survey carried out with young consumers of hallucinogenic plants and mushrooms. 2.3. Calibration standards and quality control samples Stock standard solutions (1 mg/ml) were prepared in methyl alcohol and stored at −20 ◦ C. From stock solutions, working solutions of 10, 1 and 0.1 g/ml were prepared and used for the preparation of calibration curves and quality control samples. Working solutions of MES-d9 and MDPA at a concentration of 0.1 g/ml were also prepared in methyl alcohol and stored at −20 ◦ C. Calibration standards containing limit of quantification (LOQ) concentrations, 0.1, 0.5, 1.0, 5.0 and 10.0 ng analytes under investigation per mg hair were prepared daily for each analytical batch by adding suitable amounts of working solutions to 25 mg of prechecked drug-free hair pool.
285
Quality Control (QC) samples of 0.06, 4.0 and 8.5 ng analytes under investigation per mg hair were also daily prepared to be included in each analytical batch to check validation parameters (e.g. calibration, inaccuracy, imprecision, analytical recovery, etc.). 2.4. Sample preparation Aliquots of 25 mg finely cut hair samples were weighed in a glass test tube with hermetic cap. The samples were washed with two aliquots 5 ml methyl alcohol for five min and two aliquots 2.5 ml diethyl ether for other five minutes and then dried. They were then added with 10 l internal standards (MES d9 and MDPA 0.1 g/ml) and treated with 250 l M3 reagent for 1 h at 100 ◦ C in a thermoblock. Finally, the treated samples were cooled at room temperature and 100 l M3 extract were diluted with 400 l water and a volume of 10 l diluted extract was injected into the chromatographic system. 2.5. Ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) The analyses were carried out on an ultra-high pressure liquid chromatography system (Waters Acquity UHPLC, Waters Corporation, Milan, Italy) coupled with a triple quadrupole mass spectrometer (Waters Xevo TQ, Waters Corporation). Chromatographic separation was carried out on a Acquity UHPLC HSS C18 column (2.1 mm × 150 mm, 1.8 m) using a linear gradient elution with two solvents: 0.3% formic acid in acetonitrile (solvent A) and 5 mM ammonium formate pH 3 (solvent B). Solvent A was maintained at 5% for the first 0.50 min. It was increased to 55% from 0.50 to 10.00 min, then increased to 90% from 10.00 to 10.75 min, held at 90% from 10.75 to 12.85 min, and then decreased back to 5% from 12.85 to 13.00 min and held at 5% from 13.00 to 16.50 min for reequilibration. The flow rate was kept constant at 0.40 ml/min and the column temperature was set at 50 ◦ C. The separated analytes were identified and quantified with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 3 kV, desolvation temperature 600 ◦ C, source temperature 150 ◦ C, cone gas flow rate 20 l/h, desolvation gas flow rate 1000 l/h and collision gas flow rate 0.12 ml/min. Cone energy voltages, MRM transitions, and collision energy voltages were established for each analyte and the values are listed in Table 1. 2.6. Validation procedures Validation protocol applied in the present study included linearity, limits of detection (LOD) and quantification (LOQ), imprecision, inaccuracy, selectivity, carryover, matrix effect, recovery and process efficiency, as reported elsewhere [16,17]. Validation
Table 1 Ultra-performance liquid chromatography tandem mass spectrometry parameters for the multiple reaction monitoring (MRM) acquisition mode. Analytes
Retention time (min)
MRM transitions Quantification
Psilocybin Psilocin Mescaline N,N-dimethyltryptamine Salvinorin A Mescaline-d9 3,4-Methylenedioxypropylamphetamine
1.48 2.74 3.45 3.86 10.28 3.39 5.05
Confirmation
m/z
CV (V)
CE (eV)
m/z
CV (V)
CE (eV)
285.0 > 205.0 205.2 > 58.2 212.2 > 180.1 189.1 > 143.9 433.2 > 373.1 221.2 > 186.2 222.06 > 163.0
16 18 16 16 16 16 16
20 15 20 13 10 20 15
285.0 > 240.0 205.2 > 160.0 212.2 > 165.0 189.1 > 58.2 433.2 > 313.1 221.2 > 170.4 222.06 > 86.0
16 18 16 16 16 16 16
15 10 24 13 15 24 20
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289 1.48
Psilocybin m/z 285.0 > 205.0
% 0
2.74
100
Psilocin m/z 205.2 > 58.2
%
100
1.0
1.1
1.2
1.3
1.4
1.5
1.6
100
1.7
1.8
1.9
0
2.5
2.6
2.7
2.8
DMT m/z 189.1 > 143.9
% 0
Mescaline m/z 212.2 > 180.1
3.45
2.9
3.0
100
3.1
3.2
3.3
3.4
0 3.0
10.28
3.1
3.2
3.3
3.4 3.5
3.6
3.7
3.8
3.9
Salvinorin A m/z 433.2 > 373.1
%
3.86
100
%
286
3.5
3.6 3.7 3.8
3.9 4.0
4.1 4.2
4.3 4.4
0
10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9
Fig. 1. UHPLC–MS/MS chromatogram of 25 mg drug-free hair pool spiked with 0.1 ng analytes under investigation per mg hair.
parameters were calculated from five different daily replicates of quality control samples along five subsequent working days. Linearity was determined by least-squares regression with 1/2× weighting. Acceptable linearity was achieved when the coefficient of determination was at least 0.99 and the calibrators were quantified within ±20% at the LOQ and ±15% at other concentrations. The LOD and LOQ were evaluated with decreasing analyte concentrations in drug-spiked hair samples. The LOD was defined as the lowest concentration with acceptable chromatography, the presence of all transitions with signal-to-noise ratios of at least 3, and a retention time within ±0.2 min of the average retention time of the calibrator. LOQ was the lowest concentration that met LOD criteria and a signal-to-noise ratio of at least 10. Imprecision and inaccuracy were determined at the three QC samples concentrations by analyzing five replicates on three different days. Imprecision and inaccuracy, expressed as the coefficient of variation% of the measured values and error% respectively, were expected to be less than 20%. Selectivity tests were performed with obtained from 30 healthy individuals extracted and analyzed for assessment of potential interferences from endogenous substances. Furthermore, potential interferences from principal drugs of abuse such as opiates, cocaine and metabolites, cannabinoids, principal amphetamines and methylendioxyderivatives and ketamine, were also evaluated spiking 20 mg of blank hair with 200 ng of the aforementioned substances (corresponding to 10 ng/mg hair, the highest point of calibration curve) and carried through the entire procedure. The potential for carryover was investigated by injecting extracted blank hair pool sample, with added IS, immediately after
analysis of the highest concentration point of the calibration curve on each of the days of the validation protocol and measuring the area of eventual peaks, present at the retention times of analytes under investigation. Matrix effects, recovery and process efficiency were determined using the experimental design proposed by Matuszewski et al. [18]. Set 1 were five replicates of quality control material prepared in the mobile phase. Set 2 and 3 were five replicates of blank hair samples fortified with quality control material after and before extraction, respectively. Matrix effects were determined by dividing mean peak areas of set 2 by set 1 multiplied by 100. Recovery was determined by comparing the mean peak areas of analytes under investigation obtained in set 3 to those in set 2 multiplied by 100. Process efficiency expressed as the ratio of the mean peak area of an analyte spiked before extraction (set 3) to the mean peak area of the same analyte standards (set 1) multiplied by 100. 3. Results and discussion 3.1. Chromatography and validation parameters A representative ion chromatogram obtained following the extraction of 25 mg drug-free hair pool spiked with 0.1 ng analytes under investigation per mg hair is shown in Fig. 1. Chromatographic separation was set up using the mobile phase gradient program and flow rate already optimized for the determination of principal drugs of abuse in hair samples [17]. This was made in cooperation with instrument manufacturer (Waters) choosing the best chromatographic column and mobile phase which could, in the
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
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Table 2 Calibrations results, limits of detection (LOD) and limits of quantification (LOQ) for analytes under investigation. Analyte
Slopea
Psilocybin Psilocin Mescaline N,N-dimethyltryptamine Salvinorin A
7.400 3.536 1.140 7.970 2.807
a
Intercepta ± ± ± ± ±
−0.416 −0.527 −0.066 −0.034 −0.026
3.109 0.223 0.009 3.050 1.220
± ± ± ± ±
Correlation coefficient (r2 )a 0.342 0.114 0.048 0.210 0.100
0.998 0.995 0.999 0.997 0.997
± ± ± ± ±
0.002 0.004 0.001 0.003 0.002
LOD (ng/mg hair)
LOQ (ng/mg hair)
0.01 0.01 0.02 0.01 0.02
0.04 0.04 0.05 0.03 0.05
Mean ± standard deviation of four replicates.
Table 3 Intra-day (n = 5) and inter-day (n = 15) imprecision and inaccuracy for analytes under investigation. Intra-day imprecision (coefficient ofIntra-day inaccuracy (Error%) variation%) in quality control hair in quality control hair samples samples
Analyte
Inter-day imprecision (coefficient ofInter-day inaccuracy (Error%) variation%) in quality control hair in quality control hair samples samples
0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg 0.06 ng/mg 4.0 ng/mg 8.5 ng/mg Psilocybin Psilocin Mescaline N,Ndimethyltryptamine Salvinorin A
6.8 9.1 8.1 3.1
8.6 10.6 4.5 5.9
8.9 9.7 1.7 6.0
10.3 10.7 6.2 5.9
10.6 7.6 9.9 7.7
7.3 7.3 1.6 2.1
10.2 11.2 5.2 9.8
9.8 11.0 6.1 5.0
10.2 7.8 7.9 5.0
10.9 8.5 0.1 2.0
11.1 8.5 6.0 2.1
9.5 6.1 2.9 9.0
8.8
11.7
6.3
1.9
0.2
6.1
3.8
6.6
3.0
0.5
2.8
1.2
shortest possible run-time, separate and identify as many as possible psychoactive drugs and/or metabolites in different biological matrices. Linear calibration curves were obtained for the analytes under investigation with correlation coefficients (r2 ) always better than 0.99 in all cases and LODs and LOQs values were adequate for the purpose of the present study (Table 2). The intra- and inter-assay imprecision (measured as coefficient of variation, CV %) and inaccuracy (measured as percentage error) values were always lower than 12% (Table 3). No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. As reported in Table 4, matrix effect ranged from 79.3% to 99.2%, recovery from 79.6% to 97.4%, and process efficiency from 76.1% to 99.4%. 3.2. Analysis of hair samples In order to demonstrate its applicability on real samples, the method has been used to analyze hair samples obtained from obtained by Pediatric Department of Hospital del Mar, Barcelona, Spain within the framework of a survey carried out with young consumers of hallucinogenic plants and mushrooms. Fig. 2 shows the representative ion chromatograms of 5 different hair samples of users of these psychedelic vegetal material containing all the analytes under investigation.
Just in case of a female and a male peyote consumers, segmental hair analysis could be performed since hair strands were 6 cm in both cases. In female’s hair 0.08 and 0.11 mg mescaline per mg hair was found in the two first subsequent 2 cm segments, while the compound was absent in the third 2 cm segments. In case of male consumer, 0.12, 0.13 and 0.11 mg mescaline per mg hair were measured in the three 2 cm hair segments. In all the other cases, hair samples were obtained by male consumers with short hair (from 1.5 to 2.5 cm length) and segmental hair analysis could be not performed. In the single analyzed hair shaft, 3.2 ng salvinorin A per mg hair were found in the shaft from a S. divinorum smoker, 5.6 ng N,Ndimethyltriptamine per mg hair in the shaft from an Ayahuasca user and finally 0.8 ng psilocybin per ng hair in the shaft of a psilocybe consumer. No psilocin was found in this latter sample. Concerning segmental hair analysis of mescaline in the two samples coming from peyote consumers, past repeated consumption of cacti can confirmed, which in case of female dated in the last four months prior to analysis, in case of male extended to the last 6 months. Differently from our found concentrations varying between 0.08 and 0.13 ng mescaline per mg hair, Gambelunghe et al. reported a concentration of 1 ng/mg of mescaline in the first 2 cm shaft nearest to the scalp. The difference with our results can be hypothesized in the fact that in that case the subject consumed peyote tea while in our case the two consumers smoked dried cacti. No other information exist regarding the other hallucinogenic drugs included in our study. The analysis of other hair samples is a confirmation that the psychoactive principles N,Ndimethyltryptamine, psilocin, psilocybin, salvinorin A, can be found in the hair of Ayahuasca brew psilocybe mushrooms and
Table 4 Matrix effect, recovery and process efficiency data for analytes under investigation. Recovery (%) in quality control hair samples
Process efficiency (%) in quality control hair samples
Analyte
Matrix effect (%) in quality control hair samples 0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
0.06 ng/mg
4.0 ng/mg
8.5 ng/mg
Psilocybin Psilocin Mescaline DMT Salvinorin A
94.1 92.1 92.1 89.7 79.3
89.3 91.7 99.2 87.1 80.6
90.3 92.8 89.5 89.9 89.9
95.0 90.0 94.3 84.9 97.4
88.7 94.1 89.6 80.1 80.6
84.1 84.1 85.3 85.2 79.6
86.8 81.5 87.0 98.8 83.3
84.7 79.7 89.5 99.4 85.7
81.2 84.9 76.1 97.6 87.4
288
S. Pichini et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 284–289
A 100
Psilocybin m/z 285.0 > 205.0
B 100
3.45
Mescaline m/z 212.2 > 180.1
%
%
1.48
3.85
0
0
1.6 1.7 1.8 1.9 DMT m/z 189.1 > 143.9
%
C 100
1.0 1.1 1.2 1.3 1.4
D
100
3.1
3.2
3.3
3.4
3.5
10.28
3.6
3.7
Salvinorin A m/z 433.2 > 373.1
%
0
3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4
0
10.1
10.3
10.5
10.7
10.0
Fig. 2. UHPLC–MS/MS chromatograms of 5 real hair samples of psychedelic plants and mushrooms consumers containing: (A) 0.8 ng psilocybin per mg hair; (B) 0.13 ng mescaline per mg hair; (C) 5.6 ng N,N-dimethyltryptamine per mg hair; (D) 3.2 ng salvinorin A per mg hair.
S. divinorum leaves consumers, respectively, evidencing a past consumption. No other conclusions can be drawn since these are preliminary results in a single short hair shafts. 4. Conclusion We have developed and validated an ultra-high-pressure liquid chromatography tandem mass spectrometry analysis of mescaline, N,N-dimethyltryptamine, psilocin, psilocybin, salvinorin A in hair of consumers of psychedelic vegetal material such peyote or trichocereus cacti, psilocybe mushrooms, S. divinorum or psychedelic beverage ayahuasca. applying a rapid digestion of the keratin matrix with M3 reagent. The method was tested for its feasibility in real samples and provided excellent results for determination of hallucinogenic drugs in hair of psychedelic plants and mushrooms consumers. Acknowledgements The authors thank Laura Martucci and Patrizia Gori from Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy for technical help. References [1] S.L. Hill, S.H. Thomas, Clinical toxicology of newer recreational drugs, Clin. Toxicol. (Phila) 49 (2011) 705–719, http://dx.doi.org/10.3109/ 15563650.2011.615318.
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