Forensic Science International 237 (2014) 1–6
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Synthesis, hydrolysis and stability of psilocin glucuronide Rafaela Martin a,*, Jennifer Schu¨renkamp a, Heidi Pfeiffer a, Matthias Lehr b, Helga Ko¨hler a a b
Institute of Legal Medicine, University Hospital Mu¨nster, Ro¨ntgenstr. 23, D-48149 Mu¨nster, Germany Institute of Pharmaceutical and Medicinal Chemistry, University of Mu¨nster, Corrensstr. 48, D-48149 Mu¨nster, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 July 2013 Received in revised form 15 December 2013 Accepted 6 January 2014 Available online 15 January 2014
A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4tri-O-isobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for b-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Psilocin glucuronide Synthesis Stability Hydrolysis b-Glucuronidase
1. Introduction Psilocybin and psilocin are the psychoactive substances of certain mushroom species, like Panaeolus, Conocybe and Psilocybe, which are also called ‘‘magic mushrooms’’. Their consumption causes hallucinations [1] why they are consumed as recreational drugs. Therefore they are illegal in a lot of countries [2]. In the body psilocybin is dephosphorylated rapidly to psilocin by phosphatase [3]. Uridine 50 -diphospho (UDP)-glucuronosyltransferases form psilocin glucuronide (PCG, Scheme 1), which is the main metabolite [4]. Up to now PCG is commercially not available. In literature the preparation of PCG has been described only once. It was synthesized enzymatically with rat liver microsomes and UDPglucuronic acid as a cofactor [5]. We present an alternative access to PCG by chemical synthesis. With the synthesized PCG it was possible to optimize the hydrolysis conditions of this substance. Furthermore the stability of PCG in urine, serum and whole blood was analyzed systematically. Up to now there is no data available about the stability of PCG. That way the stability of the very labile psilocin [6–11] and its metabolite PCG could be compared to gain knowledge about the
* Corresponding author. Tel.: +49 251 8355612; fax: +49 251 8355158. E-mail addresses: [email protected] (R. Martin), [email protected] (J. Schu¨renkamp), [email protected] (H. Pfeiffer), [email protected] (M. Lehr), [email protected] (H. Ko¨hler). 0379-0738/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2014.01.006
best approach to analyze samples if there is the suspicion of magic mushroom consumption. 2. Material and methods 2.1. Chemicals and reagents Methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-a-Dglucopyranuronate was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). BF3-Et2O was from Alfa Aesar (Karlsruhe, Germany). Psilocin and psilocin-d10 as reference standards were obtained as solids from Cerilliant (Round Rock, Texas, USA). The necessary amount of psilocin for the synthesis of PCG was synthesized in a modified manner according to published procedures [12–14]. b-Glucuronidase type IX-A from Escherichia coli (lyophilized powder) and b-glucuronidase type HP-2 from Helix pomatia (aqueous solution) were from Sigma–Aldrich (Steinheim, Germany). Drug-free urine, serum and blood were given by the authors. 2.2. Instrumentation Semi-preparative high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-6A pump with a Kromasil 100-5 C18 column (250 10 mm, CS Chromatographie Service) protected with an analogously filled guard column (50 10 mm) and a LCD 500 UV detector (Gamma Analysentechnik GmbH). 1H NMR spectra were recorded on a Mercury Plus 400 (400 MHz, Varian) and a DD2 600 (600 MHz, Agilent). Mass
2
R. Martin et al. / Forensic Science International 237 (2014) 1–6
Scheme 1. Pathway of psilocin glucuronide synthesis.
spectra of the synthesized substances with electron impact at 70 eV were obtained with a GCQ (Thermo Finnigan) and highresolution mass spectra with a microTOF (Bruker Daltonik). Purity of psilocin glucuronide was analyzed with a Series 1100 HPLC-DAD (Hewlett Packard) at 219, 254 and 270 nm. For LC-MS/MS conditions see [15]. 2.3. Synthesis of psilocin glucuronide 2.3.1. Methyl 1-O-[3-(2-dimethylaminoethyl)indol-4-yl]-2,3,4-Otriisobutyrylglucopyranuronate 3 Psilocin 1 (36.2 mg, 0.178 mmol) and methyl 2,3,4-tri-Oisobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate 2 (100 mg, 0.178 mmol) were stirred in 4 mL anhydrous methylene chloride in an ice bath under nitrogen (Scheme 1). BF3-Et2O (66.8 mL, 0.553 mmol) was added and the solution stirred for 24 h. After dilution with methylene chloride (4 mL) the solution was washed with saturated aqueous sodium bicarbonate, water and brine (8 mL each). The organic phase was dried and evaporated in vacuo. Flash chromatography on silica (elution with methylene chloride/methanol/ammonia 90 + 10 + 2, v/v) provided 3 as a brown solid (26.4 mg, 0.0436 mmol, 25%). 1 H NMR (400 MHz, CDCl3): d = 1.04–1.15 (m, 18H, 3 CH(CH3)2), 2.17 (s, 6H, N(CH3)2), 2.48–2.57 (m, 3H, 3 CH(CH3)2), 3.25–3.49 (m, 4H, 2 H-10 and 2 H-20 ), 3.67 (s, 3H, OCH3), 4.18 (d, 3 J = 9.7 Hz, 1H, H-500 ), 5.27 (t, 3J = 9.5 Hz, 1H, H-300 ), 5.33 (dd, 3J = 9.8, 7.9 Hz, 1H, H-200 ), 5.46–5.53 (m, 2H, H-100 and H-400 ), 6.60 (d, 3 J = 7.0 Hz, 1H, H-5), 7.07–7.14 (m, 2H, H-6 and H-7), 7.18–7.20 (m, 1H, H-2), 8.33 (s, 1H, H-1). MS (EI, 70 eV) m/z: 604 M+. 2.3.2. Psilocin glucuronide 4 3 (26 mg, 0.0430 mmol) was stirred with methanol (5 mL) and 1.5 M sodium hydroxide (0.45 mL) for 51 h at room temperature. Glacial acetic acid (60 mL) was added and the organic solvent evaporated in vacuo. Semi-preparative HPLC on a C18 column with methanol/water/formic acid (90 + 10 + 0.05, v/v) and lyophilization resulted in psilocin glucuronide 4 as a white solid (12 mg, 0.0316 mmol, 74%). Purity 98–100%. 1H NMR (600 MHz, D2O): d = 2.90 (d, 3 J = 9.8 Hz, 6H, N(CH3)2), 3.27–3.34 (m, 1H, H-20 ), 3.35–3.41 (m, 1H, H-20 ), 3.50–3.61 (m, 2H, 2 H-10 ), 3.64–3.73 (m, 3H, H200 , H-300 and H-400 ), 3.95 (d, 3J = 9.5 Hz, 1H, H-500 ), 5.25 (d, 3 J = 7.3, 1H, H-100 ), 6.86 (d, 3J = 7.7 Hz, 1H, H-5), 7.20 (t, 3 J = 8.0 Hz, 1H, H-6), 7.24–7.30 (m, 2H, H-2 and H-7), 8.44 (s, 1H, H-1). HRMS (ESI) calcd for C18H25N2O7 [M+H]+ 381.1656, found 381.1633
2.4. Hydrolysis of psilocin glucuronide For enzymatic hydrolysis of PCG the source of enzyme, the addition of ascorbic acid, the amount of enzyme and the incubation time were varied. After incubation the samples were extracted with a recently published solid phase extraction (SPE) method and the extracts measured by LC–MS/MS [15]. The area ratio of the liberated psilocin and the internal standard (IS) psilocin-d10 in the different samples were compared. 2.4.1. Comparison of b-glucuronidases from H. pomatia and E. coli For comparison of the activity of b-glucuronidases from H. pomatia and E. coli 0.5 mL urine spiked with 931 ng/mL PCG (i500 ng/mL psilocin) were diluted with 1 mL sodium acetate buffer (0.1 M; pH 4.5) in case of the H. pomatia samples and with 2 mL phosphate buffer (0.1 M; pH 4.5) in case of the E. coli samples. After addition of the appropriate enzyme (3000 U/mL each), the samples were incubated for 4 h at 37 8C. 2.4.2. Addition of ascorbic acid Urine samples were diluted with 2 mL phosphate buffer and spiked with PCG and psilocin, respectively, at a high (931 and 500 ng/mL respectively) and a low concentration (93.1 and 50 ng/ mL respectively). One set of samples was incubated with 3000 U/ mL b-glucuronidase from E. coli at 37 8C for 4 h with 10 mL 0.1 M ascorbic acid and the other one without ascorbic acid. Additionally serum samples diluted with 2 mL phosphate buffer and spiked with PCG (46.5 ng/mL i 25 ng/mL psilocin) and psilocin (25 ng/mL) respectively were incubated with and without addition of 10 mL 0.1 M ascorbic acid. 2.4.3. Incubation time and amount of enzyme Urine samples diluted with 2 mL phosphate buffer and spiked with 931 ng/mL PCG were incubated with 1000, 2000 and 3000 U/ mL b-glucuronidase from E. coli for 1, 2, 3, 4 and 15 h at 37 8C. 2.4.4. Optimal hydrolysis conditions Urine or serum samples were diluted with 2 mL phosphate buffer (0.1 M, pH 6). 2000 U/mL b-glucuronidase from E. coli and 10 mL 0.1 M ascorbic acid were added and the samples were incubated at 37 8C for 2 h. 2.5. Stability studies 2.5.1. Freeze/thaw stability and long-term stability Freeze/thaw and long-term stability of PCG and psilocin in serum and urine at two concentrations (Table 1) were evaluated
R. Martin et al. / Forensic Science International 237 (2014) 1–6
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Table 1 Freeze/thaw and long-term stability of psilocin and PCG in serum and urine; CI confidence interval. Analyte (matrix)
Concentration
Freeze/thaw stability % of controls
Psilocina,c PCGa Psilocinb PCGb
b c
93.2 94.6 94.3 106.0 98.5 91.1 101.5 100.1
90% CI
91.3–95.1 93.6-95.6 91.0–97.7 102.8–109.3 95.8–101.2 88.6–93.5 99.8–103.1 96.9–103.3
6 weeks
6 months
% of controls
90% CI
% of controls
90% CI
97.7 99.1 94.9 106.5 95.2 91.7 99.7 101.0
95.9–99.4 97.3–101.0 89.6–100.1 102.3–110.6 91.7–98.7 90.3–93.1 97.9–101.5 95.5–106.4
89.6 74.6 99.7 97.0 77.7 81.2 94.0 94.9
88.9–90.3 73.6–75.6 92.7–106.7 94.8–99.2 76.5–78.9 79.2–83.3 89.6–98.3 92.6–97.2
In serum. In urine. The data on the freeze/thaw and long-term stability of psilocin in serum have already been published [15].
according to the guidelines of the ‘‘Society of Toxicological and Forensic Chemistry’’ (GTFCh) for method validation [16]. Samples were hydrolyzed in sixfold under the optimized conditions described above, extracted and measured with the SPE-LC–MS/ MS procedure published recently [15]. Freeze/thaw stability was evaluated after three freeze/thaw cycles and long-term stability after sample storage at 20 8C for six weeks and six months. Control samples were analyzed directly without treatment. The mean values of the stability samples were compared with those of the control samples. 2.5.2. Short-term stability Short-term stability of PCG in whole blood over a period of 12 h to 7 days at room temperature (23 8C), in the fridge (4 8C) and deep frozen (20 8C) was evaluated according to the procedure described for psilocin [11]. 3. Results and discussion 3.1. Synthesis of psilocin glucuronide So far only one successful way of synthesizing psilocin glucuronide has been described. Shoda et al. used an enzymatic approach with UDP-glucuronosyltransferase [5]. The production of PCG with Koenigs–Knorr synthesis and the Williamson–Ether synthesis did not work [5]. Brown et al. reported on the chemical synthesis of paracetamol glucuronide [17] and morphine diglucuronide [18]. The PCG synthesis described in this paper was done in analogy. Psilocin 1 was reacted equimolar with methyl 2,3,4-triO-isobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate 2 in the presence of BF3-Et2O to methyl 1-O-[3-(2-dimethylaminoethyl)indol-4-yl]-2,3,4-O-triisobutyryl-glucopyranuronat 3 in 25% yield (Scheme 1). Alkaline hydrolysis gave psilocin glucuronide (PCG) 4 as a white solid in 74% yield, resulting in an overall yield of 18%. The observed mass of 4 (m/z 381.1633) obtained with highresolution MS was in accordance to the calculated mass of [M+H]+ (m/z 381.1656). 1H NMR data also confirmed the structure of 4. The coupling constant 3J = 7.3 Hz at 5.25 ppm of the anomeric proton identified 4 as the pure b-anomer which is the natural configuration of glucuronides [19]. Purity of 4 determined by HPLC–UV with different columns and mobile phases at three different wavelengths was 98–100%. The yield of the chemical synthesis presented (12 mg, 18%) was comparable to the one obtained by Shoda et al. with their enzymatic route (3.6 mg, 19%) [5]. Since it is easier to scale-up a chemical synthesis than an enzymatic one, the described procedure provides the option to obtain higher amounts of PCG.
3.2. Hydrolysis of psilocin glucuronide Optimal conditions for enzymatic hydrolysis of psilocin glucuronide in urine and serum were investigated systematically with blank samples spiked with PCG. First b-glucuronidases from E. coli and H. pomatia were compared for their effectiveness to cleave PCG in urine. The incubations were carried out at optimal temperature (37 8C) and pH values (6 and 4.5 respectively) of the enzymes. E. coli bglucuronidase showed much better results than b-glucuronidase from H. pomatia as the amount of free psilocin after hydrolysis was about three times higher with the E. coli than with the H. pomatia enzyme (Fig. 1). This is in accordance with literature data obtained by analyzing positive urine of ‘‘magic mushroom’’ users [20,21]. Enzyme preparations from H. pomatia contain also a certain sulfatase activity whereas those from E. coli are free of it [22,23]. Therefore often b-glucuronidase from H. pomatia is used, when drugs that also form sulfate conjugates are analyzed. Furthermore, the preparation from H. pomatia is cheaper and easier to handle as it can be stored refrigerated whereas the enzyme from E. coli has to be kept deep frozen [22,23]. Nevertheless the use of b-glucuronidase from E. coli is more suitable when analyzing samples for psilocin. In a second step it was analyzed, whether protection of PCG and free psilocin by ascorbic acid during incubation of urine and serum is necessary. Urine was spiked with PCG and free psilocin at two concentrations with and without addition of ascorbic acid prior to incubation. There was no difference between the samples with and
2.0
Area ratio [Psilocin/IS]
a
1 ng/mL 17.5 ng/mL 1.86 ng/mL 32.6 ng/mL 25 ng/mL 500 ng/mL 46.6 ng/mL 931 ng/mL
Long-term stability (20 8C)
1.5
1.0
0.5
0.0 E. coli
H. pomatia
Fig. 1. Comparison of PCG hydrolysis (931 ng/mL) in urine with b-glucuronidase (3000 U/mL) from E. coli and H. pomatia for 4 h at 37 8C; n = 4; bars = standard deviation.
R. Martin et al. / Forensic Science International 237 (2014) 1–6
2.0
2.5
1.5
2.0
1.0
with AA without AA
0.5
Area ratio [Psilocin/IS]
Area ratio [Psilocin/IS]
4
1.5 with AA 1.0
without AA
0.5
0.0 931 ng/mL PCG
93.1 ng/mL 500 ng/mL PCG Psilocin
50 ng/mL Psilocin
Fig. 2. Incubation of urine samples spiked at two concentration levels with PCG and psilocin with and without addition of ascorbic acid (AA); incubation with 3000 U/ mL b-glucuronidase from E. coli for 4 h at 37 8C; n = 4; bars = standard deviation.
without ascorbic acid (Fig. 2). This shows that PCG as well as psilocin are stable during incubation of urine. In contrast, in case of serum samples the addition of ascorbic acid is necessary to achieve optimal results. In absence of ascorbic acid, after the incubation of PCG spiked samples with bglucuronidase the psilocin concentration was only about one third in comparison to samples with ascorbic acid protection (Fig. 3). Furthermore, the psilocin concentration in serum samples spiked with psilocin was reduced by 50% without addition of ascorbic acid. This is in line with the findings of Kamata et al. who registered a total loss of PCG and psilocin in an authentic serum sample after incubation without ascorbic acid protection [24]. They were able to stabilize the analytes with ascorbic acid. The group did not, however, analyze the effect in urine samples. The time and amount of enzyme required for a complete cleavage was analyzed in urine at a high PCG concentration (931 ng/mL). The amount of enzyme was varied from 1000 to 3000 U/mL and the duration of incubation from 1 to 4 h. An additional incubation was done overnight (15 h). 2000 U/mL b-glucuronidase from E. coli and an incubation time of 2 h proved to be sufficient (Fig. 4). While the psilocin concentration with 2000 U/mL was constant from 2 to 4 h, it was significantly reduced after 15 h of incubation (p < 0.05 with two-tailed two-sample Student’s t-test, level of significance 95%). Therefore, the incubation time should not exceed 4 h. It was reported that for hydrolysis of 6.6 mg/mL PCG in an authentic urine sample and incubation time of 2 h with 5000 U/mL enzyme or 4 h with 3000 U/mL enzyme were necessary. However, this PCG concentration was unusually high [21].
0.0 46.5 ng/mL 25 ng/mL PCG Psilocin Fig. 3. Incubation of serum samples spiked with PCG and psilocin with and without addition of ascorbic acid (AA); incubation with 3000 U/mL b-glucuronidase from E. coli for 4 h at 37 8C; n = 4; bars = standard deviation.
In conclusion, optimal hydrolysis of PCG in urine and plasma can be achieved with 2000 U/mL b-glucuronidase from E. coli in presence of ascorbic acid (10 mL of a 0.1 M solution) applying an incubation time of 2 h at 37 8C. Under these conditions the complete cleavage of a high PCG concentration (931 ng/mL) was verified by directly measuring the sample after incubation with LCMS/MS for remaining PCG. 3.3. Stability studies Psilocin is a very unstable analyte [6–11]. In PCG the hydroxyl group is protected by glucuronic acid and therefore PCG is possibly more stable than psilocin. With the synthesized PCG it was possible to compare the stability of psilocin and PCG in different matrices under different conditions. The concentrations in the samples spiked with psilocin corresponded to the amounts of psilocin that could be liberated from the PCG samples. 3.3.1. Freeze/thaw stability and long-term stability Freeze/thaw and long-term stability were determined in serum and urine, i.e., in the matrices that are actually stored, probably thawed several times and finally analyzed. According to the GTFCh guidelines [16] stability is assumed if the mean of the stability samples is within 90–110% of the control samples and the 90% confidence interval is within 80–120%. Psilocin as well as PCG was stable after three freeze/thaw cycles
Fig. 4. Incubation of urine samples spiked with PCG (931 ng/mL) with different amounts of enzyme and incubation times; n = 4; bars = standard deviation.
a
120
% of control samples
R. Martin et al. / Forensic Science International 237 (2014) 1–6
100
5
80 room temperature, 23°C 60
fridge, 4°C deep frozen, -20°C
40 20 0 0
2
4
6
8
b
140
% of control samples
days stored
120 room temperature, 23°C 100 fridge, 4°C 80
deep frozen, -20°C
60 40 20 0 0
2
4
6
8
days stored Fig. 5. Mean area ratios of stability samples divided by the ones of control samples (percent) at different temperatures plotted vs. the storage time; n = 3; bars = standard deviations; a psilocin (50 ng/g; data from [11]); b PCG (93 ng/g).
3.3.2. Short-term stability The short-term stability (12 h to 7 days) of PCG in whole blood (with sodium fluoride as a stabilizer) under different storage conditions (room temperature, 23 8C; fridge, 4 8C; deep frozen, 20 8C) was investigated. Whole blood is the matrix that is transported from the location of sample collection to the laboratory where it is centrifuged and the supernatant is separated. The shortterm stability of psilocin in whole blood has already been discussed [11]. As the PCG standard was available now, the experiment was repeated with PCG, so both analytes could be compared. The stability samples under the different conditions were compared with control samples that had been extracted directly. PCG proved to be more stable at room temperature than psilocin as after one week still 77% PCG were present but only 12% psilocin (Fig. 5). In blood samples stored at 20 8C PCG was also more stable and had a much lower standard deviation. The stability in refrigerated samples was comparable.
quantities of product. The obtained amount of 12 mg glucuronide, however, was sufficient for characterization by 1H NMR and highresolution MS as well as to conduct several studies with PCG. Hydrolysis conditions of PCG were optimized. b-Glucuronidase from E. coli was more suitable than the enzyme from H. pomatia. Ascorbic acid should be added to serum samples to protect psilocin during incubation. The amount of enzyme and the incubation time necessary for complete cleavage were analyzed. Furthermore, the stability of psilocin and PCG was compared as stability data are the basis for forensic interpretation of measurements. Both analytes are stable in serum and urine after three freeze/thaw cycles and deep frozen at 20 8C for six weeks. PCG shows long-term stability even after six months whereas psilocin is not stable over this period. Analysis of short-term stability in whole blood for up to one week at different storage temperatures showed that PCG is clearly more stable at room temperature than psilocin. In refrigerated samples stability was comparable. Since PCG is obviously more stable than psilocin, an enzymatic hydrolysis of PCG should always be included in the analysis of psilocin in serum and urine. By that way it may still be possible to detect psilocin and thus prove magic mushroom consumption even if free psilocin has already decomposed.
4. Conclusions
References
and after storage for six weeks at 20 8C both in serum and urine (Table 1). Psilocin did not fulfill the criteria after six months in serum and urine. PCG in contrast was stable even after six months in both matrices.
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Synthesis, hydrolysis and stability of psilocin glucuronide Rafaela Martin a,*, Jennifer Schu¨renkamp a, Heidi Pfeiffer a, Matthias Lehr b, Helga Ko¨hler a a b
Institute of Legal Medicine, University Hospital Mu¨nster, Ro¨ntgenstr. 23, D-48149 Mu¨nster, Germany Institute of Pharmaceutical and Medicinal Chemistry, University of Mu¨nster, Corrensstr. 48, D-48149 Mu¨nster, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 July 2013 Received in revised form 15 December 2013 Accepted 6 January 2014 Available online 15 January 2014
A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4tri-O-isobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for b-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Psilocin glucuronide Synthesis Stability Hydrolysis b-Glucuronidase
1. Introduction Psilocybin and psilocin are the psychoactive substances of certain mushroom species, like Panaeolus, Conocybe and Psilocybe, which are also called ‘‘magic mushrooms’’. Their consumption causes hallucinations [1] why they are consumed as recreational drugs. Therefore they are illegal in a lot of countries [2]. In the body psilocybin is dephosphorylated rapidly to psilocin by phosphatase [3]. Uridine 50 -diphospho (UDP)-glucuronosyltransferases form psilocin glucuronide (PCG, Scheme 1), which is the main metabolite [4]. Up to now PCG is commercially not available. In literature the preparation of PCG has been described only once. It was synthesized enzymatically with rat liver microsomes and UDPglucuronic acid as a cofactor [5]. We present an alternative access to PCG by chemical synthesis. With the synthesized PCG it was possible to optimize the hydrolysis conditions of this substance. Furthermore the stability of PCG in urine, serum and whole blood was analyzed systematically. Up to now there is no data available about the stability of PCG. That way the stability of the very labile psilocin [6–11] and its metabolite PCG could be compared to gain knowledge about the
* Corresponding author. Tel.: +49 251 8355612; fax: +49 251 8355158. E-mail addresses: [email protected] (R. Martin), [email protected] (J. Schu¨renkamp), [email protected] (H. Pfeiffer), [email protected] (M. Lehr), [email protected] (H. Ko¨hler). 0379-0738/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2014.01.006
best approach to analyze samples if there is the suspicion of magic mushroom consumption. 2. Material and methods 2.1. Chemicals and reagents Methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-a-Dglucopyranuronate was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). BF3-Et2O was from Alfa Aesar (Karlsruhe, Germany). Psilocin and psilocin-d10 as reference standards were obtained as solids from Cerilliant (Round Rock, Texas, USA). The necessary amount of psilocin for the synthesis of PCG was synthesized in a modified manner according to published procedures [12–14]. b-Glucuronidase type IX-A from Escherichia coli (lyophilized powder) and b-glucuronidase type HP-2 from Helix pomatia (aqueous solution) were from Sigma–Aldrich (Steinheim, Germany). Drug-free urine, serum and blood were given by the authors. 2.2. Instrumentation Semi-preparative high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-6A pump with a Kromasil 100-5 C18 column (250 10 mm, CS Chromatographie Service) protected with an analogously filled guard column (50 10 mm) and a LCD 500 UV detector (Gamma Analysentechnik GmbH). 1H NMR spectra were recorded on a Mercury Plus 400 (400 MHz, Varian) and a DD2 600 (600 MHz, Agilent). Mass
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R. Martin et al. / Forensic Science International 237 (2014) 1–6
Scheme 1. Pathway of psilocin glucuronide synthesis.
spectra of the synthesized substances with electron impact at 70 eV were obtained with a GCQ (Thermo Finnigan) and highresolution mass spectra with a microTOF (Bruker Daltonik). Purity of psilocin glucuronide was analyzed with a Series 1100 HPLC-DAD (Hewlett Packard) at 219, 254 and 270 nm. For LC-MS/MS conditions see [15]. 2.3. Synthesis of psilocin glucuronide 2.3.1. Methyl 1-O-[3-(2-dimethylaminoethyl)indol-4-yl]-2,3,4-Otriisobutyrylglucopyranuronate 3 Psilocin 1 (36.2 mg, 0.178 mmol) and methyl 2,3,4-tri-Oisobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate 2 (100 mg, 0.178 mmol) were stirred in 4 mL anhydrous methylene chloride in an ice bath under nitrogen (Scheme 1). BF3-Et2O (66.8 mL, 0.553 mmol) was added and the solution stirred for 24 h. After dilution with methylene chloride (4 mL) the solution was washed with saturated aqueous sodium bicarbonate, water and brine (8 mL each). The organic phase was dried and evaporated in vacuo. Flash chromatography on silica (elution with methylene chloride/methanol/ammonia 90 + 10 + 2, v/v) provided 3 as a brown solid (26.4 mg, 0.0436 mmol, 25%). 1 H NMR (400 MHz, CDCl3): d = 1.04–1.15 (m, 18H, 3 CH(CH3)2), 2.17 (s, 6H, N(CH3)2), 2.48–2.57 (m, 3H, 3 CH(CH3)2), 3.25–3.49 (m, 4H, 2 H-10 and 2 H-20 ), 3.67 (s, 3H, OCH3), 4.18 (d, 3 J = 9.7 Hz, 1H, H-500 ), 5.27 (t, 3J = 9.5 Hz, 1H, H-300 ), 5.33 (dd, 3J = 9.8, 7.9 Hz, 1H, H-200 ), 5.46–5.53 (m, 2H, H-100 and H-400 ), 6.60 (d, 3 J = 7.0 Hz, 1H, H-5), 7.07–7.14 (m, 2H, H-6 and H-7), 7.18–7.20 (m, 1H, H-2), 8.33 (s, 1H, H-1). MS (EI, 70 eV) m/z: 604 M+. 2.3.2. Psilocin glucuronide 4 3 (26 mg, 0.0430 mmol) was stirred with methanol (5 mL) and 1.5 M sodium hydroxide (0.45 mL) for 51 h at room temperature. Glacial acetic acid (60 mL) was added and the organic solvent evaporated in vacuo. Semi-preparative HPLC on a C18 column with methanol/water/formic acid (90 + 10 + 0.05, v/v) and lyophilization resulted in psilocin glucuronide 4 as a white solid (12 mg, 0.0316 mmol, 74%). Purity 98–100%. 1H NMR (600 MHz, D2O): d = 2.90 (d, 3 J = 9.8 Hz, 6H, N(CH3)2), 3.27–3.34 (m, 1H, H-20 ), 3.35–3.41 (m, 1H, H-20 ), 3.50–3.61 (m, 2H, 2 H-10 ), 3.64–3.73 (m, 3H, H200 , H-300 and H-400 ), 3.95 (d, 3J = 9.5 Hz, 1H, H-500 ), 5.25 (d, 3 J = 7.3, 1H, H-100 ), 6.86 (d, 3J = 7.7 Hz, 1H, H-5), 7.20 (t, 3 J = 8.0 Hz, 1H, H-6), 7.24–7.30 (m, 2H, H-2 and H-7), 8.44 (s, 1H, H-1). HRMS (ESI) calcd for C18H25N2O7 [M+H]+ 381.1656, found 381.1633
2.4. Hydrolysis of psilocin glucuronide For enzymatic hydrolysis of PCG the source of enzyme, the addition of ascorbic acid, the amount of enzyme and the incubation time were varied. After incubation the samples were extracted with a recently published solid phase extraction (SPE) method and the extracts measured by LC–MS/MS [15]. The area ratio of the liberated psilocin and the internal standard (IS) psilocin-d10 in the different samples were compared. 2.4.1. Comparison of b-glucuronidases from H. pomatia and E. coli For comparison of the activity of b-glucuronidases from H. pomatia and E. coli 0.5 mL urine spiked with 931 ng/mL PCG (i500 ng/mL psilocin) were diluted with 1 mL sodium acetate buffer (0.1 M; pH 4.5) in case of the H. pomatia samples and with 2 mL phosphate buffer (0.1 M; pH 4.5) in case of the E. coli samples. After addition of the appropriate enzyme (3000 U/mL each), the samples were incubated for 4 h at 37 8C. 2.4.2. Addition of ascorbic acid Urine samples were diluted with 2 mL phosphate buffer and spiked with PCG and psilocin, respectively, at a high (931 and 500 ng/mL respectively) and a low concentration (93.1 and 50 ng/ mL respectively). One set of samples was incubated with 3000 U/ mL b-glucuronidase from E. coli at 37 8C for 4 h with 10 mL 0.1 M ascorbic acid and the other one without ascorbic acid. Additionally serum samples diluted with 2 mL phosphate buffer and spiked with PCG (46.5 ng/mL i 25 ng/mL psilocin) and psilocin (25 ng/mL) respectively were incubated with and without addition of 10 mL 0.1 M ascorbic acid. 2.4.3. Incubation time and amount of enzyme Urine samples diluted with 2 mL phosphate buffer and spiked with 931 ng/mL PCG were incubated with 1000, 2000 and 3000 U/ mL b-glucuronidase from E. coli for 1, 2, 3, 4 and 15 h at 37 8C. 2.4.4. Optimal hydrolysis conditions Urine or serum samples were diluted with 2 mL phosphate buffer (0.1 M, pH 6). 2000 U/mL b-glucuronidase from E. coli and 10 mL 0.1 M ascorbic acid were added and the samples were incubated at 37 8C for 2 h. 2.5. Stability studies 2.5.1. Freeze/thaw stability and long-term stability Freeze/thaw and long-term stability of PCG and psilocin in serum and urine at two concentrations (Table 1) were evaluated
R. Martin et al. / Forensic Science International 237 (2014) 1–6
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Table 1 Freeze/thaw and long-term stability of psilocin and PCG in serum and urine; CI confidence interval. Analyte (matrix)
Concentration
Freeze/thaw stability % of controls
Psilocina,c PCGa Psilocinb PCGb
b c
93.2 94.6 94.3 106.0 98.5 91.1 101.5 100.1
90% CI
91.3–95.1 93.6-95.6 91.0–97.7 102.8–109.3 95.8–101.2 88.6–93.5 99.8–103.1 96.9–103.3
6 weeks
6 months
% of controls
90% CI
% of controls
90% CI
97.7 99.1 94.9 106.5 95.2 91.7 99.7 101.0
95.9–99.4 97.3–101.0 89.6–100.1 102.3–110.6 91.7–98.7 90.3–93.1 97.9–101.5 95.5–106.4
89.6 74.6 99.7 97.0 77.7 81.2 94.0 94.9
88.9–90.3 73.6–75.6 92.7–106.7 94.8–99.2 76.5–78.9 79.2–83.3 89.6–98.3 92.6–97.2
In serum. In urine. The data on the freeze/thaw and long-term stability of psilocin in serum have already been published [15].
according to the guidelines of the ‘‘Society of Toxicological and Forensic Chemistry’’ (GTFCh) for method validation [16]. Samples were hydrolyzed in sixfold under the optimized conditions described above, extracted and measured with the SPE-LC–MS/ MS procedure published recently [15]. Freeze/thaw stability was evaluated after three freeze/thaw cycles and long-term stability after sample storage at 20 8C for six weeks and six months. Control samples were analyzed directly without treatment. The mean values of the stability samples were compared with those of the control samples. 2.5.2. Short-term stability Short-term stability of PCG in whole blood over a period of 12 h to 7 days at room temperature (23 8C), in the fridge (4 8C) and deep frozen (20 8C) was evaluated according to the procedure described for psilocin [11]. 3. Results and discussion 3.1. Synthesis of psilocin glucuronide So far only one successful way of synthesizing psilocin glucuronide has been described. Shoda et al. used an enzymatic approach with UDP-glucuronosyltransferase [5]. The production of PCG with Koenigs–Knorr synthesis and the Williamson–Ether synthesis did not work [5]. Brown et al. reported on the chemical synthesis of paracetamol glucuronide [17] and morphine diglucuronide [18]. The PCG synthesis described in this paper was done in analogy. Psilocin 1 was reacted equimolar with methyl 2,3,4-triO-isobutyryl-1-O-trichloroacetimidoyl-a-D-glucopyranuronate 2 in the presence of BF3-Et2O to methyl 1-O-[3-(2-dimethylaminoethyl)indol-4-yl]-2,3,4-O-triisobutyryl-glucopyranuronat 3 in 25% yield (Scheme 1). Alkaline hydrolysis gave psilocin glucuronide (PCG) 4 as a white solid in 74% yield, resulting in an overall yield of 18%. The observed mass of 4 (m/z 381.1633) obtained with highresolution MS was in accordance to the calculated mass of [M+H]+ (m/z 381.1656). 1H NMR data also confirmed the structure of 4. The coupling constant 3J = 7.3 Hz at 5.25 ppm of the anomeric proton identified 4 as the pure b-anomer which is the natural configuration of glucuronides [19]. Purity of 4 determined by HPLC–UV with different columns and mobile phases at three different wavelengths was 98–100%. The yield of the chemical synthesis presented (12 mg, 18%) was comparable to the one obtained by Shoda et al. with their enzymatic route (3.6 mg, 19%) [5]. Since it is easier to scale-up a chemical synthesis than an enzymatic one, the described procedure provides the option to obtain higher amounts of PCG.
3.2. Hydrolysis of psilocin glucuronide Optimal conditions for enzymatic hydrolysis of psilocin glucuronide in urine and serum were investigated systematically with blank samples spiked with PCG. First b-glucuronidases from E. coli and H. pomatia were compared for their effectiveness to cleave PCG in urine. The incubations were carried out at optimal temperature (37 8C) and pH values (6 and 4.5 respectively) of the enzymes. E. coli bglucuronidase showed much better results than b-glucuronidase from H. pomatia as the amount of free psilocin after hydrolysis was about three times higher with the E. coli than with the H. pomatia enzyme (Fig. 1). This is in accordance with literature data obtained by analyzing positive urine of ‘‘magic mushroom’’ users [20,21]. Enzyme preparations from H. pomatia contain also a certain sulfatase activity whereas those from E. coli are free of it [22,23]. Therefore often b-glucuronidase from H. pomatia is used, when drugs that also form sulfate conjugates are analyzed. Furthermore, the preparation from H. pomatia is cheaper and easier to handle as it can be stored refrigerated whereas the enzyme from E. coli has to be kept deep frozen [22,23]. Nevertheless the use of b-glucuronidase from E. coli is more suitable when analyzing samples for psilocin. In a second step it was analyzed, whether protection of PCG and free psilocin by ascorbic acid during incubation of urine and serum is necessary. Urine was spiked with PCG and free psilocin at two concentrations with and without addition of ascorbic acid prior to incubation. There was no difference between the samples with and
2.0
Area ratio [Psilocin/IS]
a
1 ng/mL 17.5 ng/mL 1.86 ng/mL 32.6 ng/mL 25 ng/mL 500 ng/mL 46.6 ng/mL 931 ng/mL
Long-term stability (20 8C)
1.5
1.0
0.5
0.0 E. coli
H. pomatia
Fig. 1. Comparison of PCG hydrolysis (931 ng/mL) in urine with b-glucuronidase (3000 U/mL) from E. coli and H. pomatia for 4 h at 37 8C; n = 4; bars = standard deviation.
R. Martin et al. / Forensic Science International 237 (2014) 1–6
2.0
2.5
1.5
2.0
1.0
with AA without AA
0.5
Area ratio [Psilocin/IS]
Area ratio [Psilocin/IS]
4
1.5 with AA 1.0
without AA
0.5
0.0 931 ng/mL PCG
93.1 ng/mL 500 ng/mL PCG Psilocin
50 ng/mL Psilocin
Fig. 2. Incubation of urine samples spiked at two concentration levels with PCG and psilocin with and without addition of ascorbic acid (AA); incubation with 3000 U/ mL b-glucuronidase from E. coli for 4 h at 37 8C; n = 4; bars = standard deviation.
without ascorbic acid (Fig. 2). This shows that PCG as well as psilocin are stable during incubation of urine. In contrast, in case of serum samples the addition of ascorbic acid is necessary to achieve optimal results. In absence of ascorbic acid, after the incubation of PCG spiked samples with bglucuronidase the psilocin concentration was only about one third in comparison to samples with ascorbic acid protection (Fig. 3). Furthermore, the psilocin concentration in serum samples spiked with psilocin was reduced by 50% without addition of ascorbic acid. This is in line with the findings of Kamata et al. who registered a total loss of PCG and psilocin in an authentic serum sample after incubation without ascorbic acid protection [24]. They were able to stabilize the analytes with ascorbic acid. The group did not, however, analyze the effect in urine samples. The time and amount of enzyme required for a complete cleavage was analyzed in urine at a high PCG concentration (931 ng/mL). The amount of enzyme was varied from 1000 to 3000 U/mL and the duration of incubation from 1 to 4 h. An additional incubation was done overnight (15 h). 2000 U/mL b-glucuronidase from E. coli and an incubation time of 2 h proved to be sufficient (Fig. 4). While the psilocin concentration with 2000 U/mL was constant from 2 to 4 h, it was significantly reduced after 15 h of incubation (p < 0.05 with two-tailed two-sample Student’s t-test, level of significance 95%). Therefore, the incubation time should not exceed 4 h. It was reported that for hydrolysis of 6.6 mg/mL PCG in an authentic urine sample and incubation time of 2 h with 5000 U/mL enzyme or 4 h with 3000 U/mL enzyme were necessary. However, this PCG concentration was unusually high [21].
0.0 46.5 ng/mL 25 ng/mL PCG Psilocin Fig. 3. Incubation of serum samples spiked with PCG and psilocin with and without addition of ascorbic acid (AA); incubation with 3000 U/mL b-glucuronidase from E. coli for 4 h at 37 8C; n = 4; bars = standard deviation.
In conclusion, optimal hydrolysis of PCG in urine and plasma can be achieved with 2000 U/mL b-glucuronidase from E. coli in presence of ascorbic acid (10 mL of a 0.1 M solution) applying an incubation time of 2 h at 37 8C. Under these conditions the complete cleavage of a high PCG concentration (931 ng/mL) was verified by directly measuring the sample after incubation with LCMS/MS for remaining PCG. 3.3. Stability studies Psilocin is a very unstable analyte [6–11]. In PCG the hydroxyl group is protected by glucuronic acid and therefore PCG is possibly more stable than psilocin. With the synthesized PCG it was possible to compare the stability of psilocin and PCG in different matrices under different conditions. The concentrations in the samples spiked with psilocin corresponded to the amounts of psilocin that could be liberated from the PCG samples. 3.3.1. Freeze/thaw stability and long-term stability Freeze/thaw and long-term stability were determined in serum and urine, i.e., in the matrices that are actually stored, probably thawed several times and finally analyzed. According to the GTFCh guidelines [16] stability is assumed if the mean of the stability samples is within 90–110% of the control samples and the 90% confidence interval is within 80–120%. Psilocin as well as PCG was stable after three freeze/thaw cycles
Fig. 4. Incubation of urine samples spiked with PCG (931 ng/mL) with different amounts of enzyme and incubation times; n = 4; bars = standard deviation.
a
120
% of control samples
R. Martin et al. / Forensic Science International 237 (2014) 1–6
100
5
80 room temperature, 23°C 60
fridge, 4°C deep frozen, -20°C
40 20 0 0
2
4
6
8
b
140
% of control samples
days stored
120 room temperature, 23°C 100 fridge, 4°C 80
deep frozen, -20°C
60 40 20 0 0
2
4
6
8
days stored Fig. 5. Mean area ratios of stability samples divided by the ones of control samples (percent) at different temperatures plotted vs. the storage time; n = 3; bars = standard deviations; a psilocin (50 ng/g; data from [11]); b PCG (93 ng/g).
3.3.2. Short-term stability The short-term stability (12 h to 7 days) of PCG in whole blood (with sodium fluoride as a stabilizer) under different storage conditions (room temperature, 23 8C; fridge, 4 8C; deep frozen, 20 8C) was investigated. Whole blood is the matrix that is transported from the location of sample collection to the laboratory where it is centrifuged and the supernatant is separated. The shortterm stability of psilocin in whole blood has already been discussed [11]. As the PCG standard was available now, the experiment was repeated with PCG, so both analytes could be compared. The stability samples under the different conditions were compared with control samples that had been extracted directly. PCG proved to be more stable at room temperature than psilocin as after one week still 77% PCG were present but only 12% psilocin (Fig. 5). In blood samples stored at 20 8C PCG was also more stable and had a much lower standard deviation. The stability in refrigerated samples was comparable.
quantities of product. The obtained amount of 12 mg glucuronide, however, was sufficient for characterization by 1H NMR and highresolution MS as well as to conduct several studies with PCG. Hydrolysis conditions of PCG were optimized. b-Glucuronidase from E. coli was more suitable than the enzyme from H. pomatia. Ascorbic acid should be added to serum samples to protect psilocin during incubation. The amount of enzyme and the incubation time necessary for complete cleavage were analyzed. Furthermore, the stability of psilocin and PCG was compared as stability data are the basis for forensic interpretation of measurements. Both analytes are stable in serum and urine after three freeze/thaw cycles and deep frozen at 20 8C for six weeks. PCG shows long-term stability even after six months whereas psilocin is not stable over this period. Analysis of short-term stability in whole blood for up to one week at different storage temperatures showed that PCG is clearly more stable at room temperature than psilocin. In refrigerated samples stability was comparable. Since PCG is obviously more stable than psilocin, an enzymatic hydrolysis of PCG should always be included in the analysis of psilocin in serum and urine. By that way it may still be possible to detect psilocin and thus prove magic mushroom consumption even if free psilocin has already decomposed.
4. Conclusions
References
and after storage for six weeks at 20 8C both in serum and urine (Table 1). Psilocin did not fulfill the criteria after six months in serum and urine. PCG in contrast was stable even after six months in both matrices.
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