Letter - application note Received: 23 March 2014
Revised: 12 June 2014
Accepted: 16 June 2014
Published online in Wiley Online Library: 20 July 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4104
Rapid determination of ibotenic acid and muscimol in human urine S. Deja,a E. Jawień,b I. Jasicka-Misiak,a M. Halama,c P. Wieczorek,a P. Kafarskib and P. Młynarzb*
Introduction
Materials and methods
Amanita muscaria and Amanita pantherina belong to a group of hallucinogenic mushrooms that contain two psychoactive substances: ibotenic acid (IBO) and muscimol (MUS). These mushrooms have been known for many years and have been used by Siberian tribes in ethnic rituals, where the urine of shaman or deer that consumed and digested the mushrooms was drunk to experience hallucinogenic stimulation.[1] Currently, there are two ways to induce mushroom intoxication: accidentally, by mistaking hallucinogenic mushrooms for eatable fungi, or deliberately, by ingesting mushrooms of the Amanita family for recreation purposes, which has become increasingly popular. The recreational use of hallucinogenic mushrooms has become popular among young people because of easy access to the psychedelic material, which is legally accessible. Mushrooms can be gathered from the forest or purchased on the Internet. Both substances, MUS and IBO, are responsible for ‘mindaltering’ effects, and IBO may be converted to MUS in the human body. These two substances have been found in urine 1 h after consumption.[1,2] Their properties include the ability to structurally mimic two endogenous neurotransmitters, glutamic acid-IBO, and gamma-aminobutyric acid - MUS.[3,4] A portion of a hallucinogenic compound used for recreation purpose can be rapidly absorbed from the gastrointestinal tract and is excreted through urine. A single active dose of hallucinogens is approximately 6 mg of muscimol (MUS) and 20-60 mg of ibotenic acid (IBO), and the greatest effects occur over 2–3 h after ingestion.[5,6] After uptake, the concentration of both psychoactive substances is relatively high in urine, which is extremely important for noninvasive and easy collection of diagnostic material. Although Amanita mushrooms are well known for their psychoactive toxic substances, there is no commercial test for either IBO or MUS.[7] Few papers in the literature have described the determination of these compounds in body fluids and mushroom fruiting bodies.[8–10] In this study, we report for the first time a method for the detection and quantification of MUS and IBO acid in human urine using 1H NMR spectroscopy. The method was performed by spiking solutions and applying a simple 1H NMR pulse sequence (1D-NOESY experiment), typically utilized in metabolomics experiments.[10] This method enables the detection of IBO or MUS, as well as the presence of other metabolites or drugs.
Sample preparation
NMR spectroscopy All NMR spectra were recorded on a Bruker Biospin Avance II NMR (Bruker, GmbH, Germany) spectrometer operating at a proton frequency of 600.58 MHz. All samples were measured using a 1D-NOESY pulse sequence because of its ability to suppress water and proton-exchange signals.[11] The 1D-NOESY pulse sequence is a gold standard in NMR-based urine metabolomics; therefore, the initial parameters were set according to typical metabolomic profiling experiments. Each spectrum consisted of 64 scans in a 20-ppm spectral width and 32 k data points. To reduce signal loss due to relaxation, the relaxation delay was set to 3.5 s, which represented a good compromise between
* Correspondence to: P. Młynarz, Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50370 Wrocław, Poland. E-mail: [email protected] a Faculty of Chemistry, Opole University, Oleska 48, 45-051, Opole, Poland b Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland c Museum of Natural History, Wrocław University, Sienkiewicza 21, 50-335, Wrocław, Poland
Copyright © 2014 John Wiley & Sons, Ltd.
711
Magn. Reson. Chem. 2014, 52, 711–714
Two types of samples were used in this study: calibration curve samples and spiked urine samples. In order to create calibration curves for MUS and IBO, 540 μl of the deionized H2O containing particular concentration of standard compound solution was mixed with 60 μl of phosphate buffer solution (PBS) containing 99.8% D2O, sodium azide, and the internal standard, deuterated 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid (TSP-d4). For spiked urine samples, freshly collected urine was split into 530 μl aliquots and placed in polypropylene Eppendorf tubes. Urine samples were mixed with 60 μl of PBS, the same as for calibration curves. Next, 10 μl of the standard compound solution was added to the sample (one at each time IBO or MUS), whereas in the control sample, it was replaced with 10 μl of the deionized H2O. All prepared samples containing standard solutions are listed in Table 1. The samples were mixed and centrifuged at 12 000 rpm for 5 min. In total, 550 μl supernatant was transferred into 5 mm NMR tubes.
S. Deja et al. Table 1. List of samples used and concentrations of standard compounds in calibration curve samples and spiked urine samples Muscimol (μg/ml) 277.78 166.67 111.11 55.56 27.78 13.89 8.33 5.56 2.78 1.39 55.56 38.89 27.78 22.22 16.67 5.56 2.78 1.94
Ibotenic acid (μg/ml) 416.67 250.00 166.67 83.33 41.67 20.83 12.50 8.33 4.17 2.08 83.33 58.33 41.67 33.33 25.00 8.33 4.17 2.92
Sample type Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine
acquisition time and quantification accuracy. Several mixing times were examined, namely 10, 100, 200, 400, 600, and 800 ms. The phase and baseline correction were performed manually. Regions on the spectra containing MUS and IBO at 5.8–5.9 ppm were utilized for signal integral calculations. Data preprocessing A Fourier transformation was performed on all free induction decays with 0.3 Hz line broadening. The phase and baseline corrections were manually conducted using Topspin 1.3 software (Bruker, GmBH, Germany). The spectra were calibrated to the TSP signal (δ = 0.0 ppm) and exported to MATLAB, where alignment of the region of interest (5.4–6.2 ppm) was performed using the icoshift algorithm.[12] The signals were integrated and expressed as raw metabolite integrals and as a ratio of raw metabolite integrals relative to the TSP signal. The calibration curves were obtained in MS Excel, where were calculated curves equations, standard deviation of the regression lines (SD), and limit of detection (LOD = 3.3(SD/slope of the curve)).
compounds, the exogenous substances that can be metabolized by humans and those that remain unchanged can be quickly identified provided they are concentrated enough.[15,16] The primary Amanita hallucinogenic compounds are IBO and MUS (Scheme 1), each of which has only two signals on the 1H NMR spectra. Aliphatic signals from the methine group and methylene protons were present; however, these signals may be obscured by urine metabolites, e.g. glucose, potential xenobiotics or residual H2O signal.[17,18] The only way to analyze both substances (IBO and MUS) was to examine resonance signals belonging to the isoxazole ring at 5.88 ppm and 5.86 (Fig. 1). Unfortunately, the compounds of interest only appeared over the urea signal, which limited accuracy and significantly lowered the detection limit. Therefore, the method to analyze the isoxazole proton on the 1D-NMR spectra among the urine matrix metabolites was developed. For this purpose, the 1D-NOESY spectra were used with a water presaturation pulse. The advantage of this pulse sequence is that protons exhibiting intermolecular exchange with water would be also attenuated during presaturation depending on the pulse sequence parameters.[15] To assess the detection limit, samples with various concentrations were measured, starting with the highest concentration and decreasing to 2–3 μg/ml. For each spectrum, various mixing times were used, which effectively suppressed the water signal and decreased the broad urea signal originating from exchangeable NH protons.[15] During mixing time optimization, the values were continuously increased from 10 to 800 ms. However, the optimal mixing time strongly depended on the urea content of the urine sample; at high signals, 600 ms was required, but at lower signals, 300–400 ms may be sufficient. Thus, 600 ms was used for all experiments.
Scheme 1. The structures of (a) ibotenic acid and (b) muscimol.
Results and discussion
712
Despite the many studies dedicated to the analysis of hallucinogenic compounds, which can be identified in biofluids after ingestion, there is a need for rapid and low-cost analytical methods to detect various compounds. This issue is especially important because of the increased popularity of the fungi and easy access to the natural not prohibited stimulants. The primary detection method is mass spectrometry, but special sample preparation procedures are needed, as well as the use of either GC or HPLC-MS tandem methods.[5,13] Because of the increasing number of available NMR spectrometers and their commercial use and hence of cost reducing of measurements, we have demonstrated the usefulness of an NMR method in the field. Approximately at least 20–40 compounds in urine of healthy donors can be characterized by NMR spectroscopy, where their analysis might be qualitative or quantitative.[14] Among these
wileyonlinelibrary.com/journal/mrc
Figure 1. Influence of increasing mixing time on the urea signal attenuation for (a) the muscimol sample (singlet at 5.86 ppm) and (b) the ibotenic acid sample (singlet at 5.88 ppm).
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 711–714
Determination of ibotenic acid and muscimol in urine
Figure 2. Effect of normalizing the signal integrity of hippuric acid, dimethylamine (DMA) and citric acid to the internal standard TSP: (a) raw spectrum and (b) spectrum after normalization.
When increasing the mixing time, some resonance signals experienced a considerable reduction of signal intensity. Some signals were not affected, e.g. hippuric acid, whereas others, such as citric acid, changed significantly (Fig. 2). The influence of mixing time on the resonance signal intensity of selected metabolites is given in Table 2. The relative standard deviation illustrates the average change among the measurements. The selected signals were normalized to TSP using the MATLAB program (Fig. 2). Because of the non-uniformity of the urine, the calibration curves (600 ms mixing time) were performed in PBS and showed linearity with R2 = 0.9996 for MUS over the range from 278 to 3 μg/ml (LOD = 13 μg/ml) and R2 = 0.9990 for IBO from 417 to 2 μg/ml (LOD = 30 μg/ml). The obtained curves with equations for MUS
y = 0.0031 × 0.0025 and IBO y = 0.0022 × 0.0044 were used to calculate the amount of MUS and IBO spiked in the urine (Fig. 3). The method showed very good precision for IBO even below LOD value down to 25.09 μg/ml (0.4 % difference), where the sample with 10 μg/ml showed a 20% deviation due to small signal-to-noise ratio (Table 3). The detection range was sufficient to cover the IBO range (32–55 μg/ml) in urine after fungi consumption.[13] However, the MUS level in human urine after fungi ingestion is much lower (10–7 μg/ml), and this method provided accuracy at 7 μg/ml with a 27% deviation, although this strongly depends on sample acquisition time, where the signal-to-noise ratio may be strongly improved.
Table 2. The influence of mixing times of 10–800 ms on the signal intensities of the selected metabolites
Table 3. The test sample concentrations of muscimol and ibotenic acid spiked in urine with % error from the calibration curve
Selected metabolites
1 2 3 4 5 6 7 8 9 10
Alanine Alanine/TSP Citric acid Citric acid/TSP Creatinine Creatinine/TSP Formic acid Formic acid/TSP Hippuric acid Hippuric acid/TSP
Magn. Reson. Chem. 2014, 52, 711–714
Muscimol
Relative standard deviation (%) 29.09 21.38 34.02 26.61 11.93 4.53 10.32 4.56 9.94 3.23
C (μg/ml) from NMR 58.26 41.81 28.81 23.68 18.68 7.06 4.84 3.48
Copyright © 2014 John Wiley & Sons, Ltd.
Ibotenic acid
% difference
C (μg/ml) from NMR
% difference
4.86 7.50 3.70 6.55 12.06 27.16 74.19 79.17
80.09 60.50 43.68 34.73 25.09 10.00 7.77 6.55
3.89 3.71 4.84 4.18 0.36 20.00 86.55 124.42
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No.
S. Deja et al.
Figure 3. Calculated calibration curves in PBS (black triangles) and overlapped spiked urine (red circles) for (a) muscimol and (b) ibotenic acid.
Conclusion In this study, a quick and inexpensive method was examined and used to analyze two common Amanita neurotoxins (MUS and IBO) spiked in urine samples. A 1D-NOESY experiment was performed on samples at various mixing times; a mixing time of 600 ms was adequate to decrease the overlapping urea signal and enabled the quantitative and qualitative analysis of the MUS and IBO compounds.
Acknowledgements The research was supported by the Wroclaw Research Centre EIT+ under the project named ‘Biotechnologies and advanced medical technologies’—BioMed (POIG.01.01.02-02-003/08) and financed by the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). Stanisław Deja was a recipient of PhD scholarship under a project funded by the European Social Fund.
References [1] D. Michelot, L. M. Melendez-Howell. Mycol. Res. 2003, 107, 131–146. doi:10.1017/S0953756203007305. [2] J. Ott, S. P. S. Wheaton, W. S. Chilton. Physiol. Chem. Phys. 1975, 7, 381–384. [3] R. J. Walker, G. N. Woodruff, G. A. Kerkut. Comp. Gen. Pharmacol. 1971, 2, 168–174.
[4] J. Stříbrný, M. Sokol, B. Merová, P. Ondra. Int. J. Legal Med. 2012, 126, 519–524. doi:10.1007/s00414-011-0599-9. [5] J. H. Halpern. Pharmacol. Ther. 2004, 102, 131–138. doi:10.1016/j. pharmthera.2004.03.003. [6] F. G. Waser, The pharmacology of Amanita muscaria, in Ethnopharmacologic Search for Psychoactive Drugs, in Ethnopharmacologic Search for Psychoactive Drugs (Eds: D. H. Efron, B. Holmstedt, N. S. Kline), U.S. Public Health Service Publication, Washington, DC, 1979, 1645, pp. 419–439. [7] C. Li, N. H. Oberlies. Life Sci. 2005, 78, 532–538. doi:10.1016/j. lfs.2005.09.003. [8] K. Hasegawa, K. Gonmori, H. Fujita, Y. Kamijo, H. Nozawa, I. Yamagishi, K. Minakata, K. Watanabe, O. Suzuki. Forensic Toxicol. 2013, 31, 322–327. doi:10.1007/s11419-013-0189-2. [9] K. Tsujikawa, K. Kuwayama, H. Miyaguchi, T. Kanamori, Y. Iwata, H. Inoue, T. Yoshida, T. Kishi. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 852, 430–435. doi:10.1016/j.jchromb.2007.01.046. [10] R. T. McKay. Concepts Magn. Reson. Part A 2011, 48A, 197–220. doi:10.1002/cmr.a.20223. [11] S. Macura, Y. Huang, D. Suter, R. R. Ernst. J. Magn. Reson. 1981, 43, 259–281. [12] F. Savorani, G. Tomasi, S. B. Engelsen. J. Magn. Reson. 2010, 202, 190–202. doi:10.1016/j.jmr.2009.11.012. [13] M. C. Gennaro, D. Giacosa, E. Gioannini, S. Angelino. J. Liq. Chromatogr. Related Technol. 1997, 20, 413–424. [14] S. Deja, E. Barg, P. Młynarz, A. Basiak, E. Willak-Janc. J. Pharm. Biomed. Anal. 2013, 83, 43–48. doi:10.1016/j.jpba.2013.04.017. [15] Q. N. Van, in Proteomic and Metabolomic Approaches to Biomarker Discovery (Eds: H. J. Issaq, T. D. Veenstra), Academic Press, London, Waltham, San Diego, 2013, pp. 87–117. [16] M. R. Meyer, J. Wilhelm, F. T. Peters, H. H. Maurer. Anal. Bioanal. Chem. 2010, 397, 1225–1233. doi:10.1007/s00216-010-3636-5. [17] K. Tsunoda, N. Inoue, Y. Aoyagi, T. Sugahara. J. Food Hyg. Soc. Jpn. 1993, 34, 12–17. [18] N. Nakamura. Chem. Pharm. Bull. 1971, 19, 46–51.
714 wileyonlinelibrary.com/journal/mrc
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 711–714
Revised: 12 June 2014
Accepted: 16 June 2014
Published online in Wiley Online Library: 20 July 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4104
Rapid determination of ibotenic acid and muscimol in human urine S. Deja,a E. Jawień,b I. Jasicka-Misiak,a M. Halama,c P. Wieczorek,a P. Kafarskib and P. Młynarzb*
Introduction
Materials and methods
Amanita muscaria and Amanita pantherina belong to a group of hallucinogenic mushrooms that contain two psychoactive substances: ibotenic acid (IBO) and muscimol (MUS). These mushrooms have been known for many years and have been used by Siberian tribes in ethnic rituals, where the urine of shaman or deer that consumed and digested the mushrooms was drunk to experience hallucinogenic stimulation.[1] Currently, there are two ways to induce mushroom intoxication: accidentally, by mistaking hallucinogenic mushrooms for eatable fungi, or deliberately, by ingesting mushrooms of the Amanita family for recreation purposes, which has become increasingly popular. The recreational use of hallucinogenic mushrooms has become popular among young people because of easy access to the psychedelic material, which is legally accessible. Mushrooms can be gathered from the forest or purchased on the Internet. Both substances, MUS and IBO, are responsible for ‘mindaltering’ effects, and IBO may be converted to MUS in the human body. These two substances have been found in urine 1 h after consumption.[1,2] Their properties include the ability to structurally mimic two endogenous neurotransmitters, glutamic acid-IBO, and gamma-aminobutyric acid - MUS.[3,4] A portion of a hallucinogenic compound used for recreation purpose can be rapidly absorbed from the gastrointestinal tract and is excreted through urine. A single active dose of hallucinogens is approximately 6 mg of muscimol (MUS) and 20-60 mg of ibotenic acid (IBO), and the greatest effects occur over 2–3 h after ingestion.[5,6] After uptake, the concentration of both psychoactive substances is relatively high in urine, which is extremely important for noninvasive and easy collection of diagnostic material. Although Amanita mushrooms are well known for their psychoactive toxic substances, there is no commercial test for either IBO or MUS.[7] Few papers in the literature have described the determination of these compounds in body fluids and mushroom fruiting bodies.[8–10] In this study, we report for the first time a method for the detection and quantification of MUS and IBO acid in human urine using 1H NMR spectroscopy. The method was performed by spiking solutions and applying a simple 1H NMR pulse sequence (1D-NOESY experiment), typically utilized in metabolomics experiments.[10] This method enables the detection of IBO or MUS, as well as the presence of other metabolites or drugs.
Sample preparation
NMR spectroscopy All NMR spectra were recorded on a Bruker Biospin Avance II NMR (Bruker, GmbH, Germany) spectrometer operating at a proton frequency of 600.58 MHz. All samples were measured using a 1D-NOESY pulse sequence because of its ability to suppress water and proton-exchange signals.[11] The 1D-NOESY pulse sequence is a gold standard in NMR-based urine metabolomics; therefore, the initial parameters were set according to typical metabolomic profiling experiments. Each spectrum consisted of 64 scans in a 20-ppm spectral width and 32 k data points. To reduce signal loss due to relaxation, the relaxation delay was set to 3.5 s, which represented a good compromise between
* Correspondence to: P. Młynarz, Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50370 Wrocław, Poland. E-mail: [email protected] a Faculty of Chemistry, Opole University, Oleska 48, 45-051, Opole, Poland b Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland c Museum of Natural History, Wrocław University, Sienkiewicza 21, 50-335, Wrocław, Poland
Copyright © 2014 John Wiley & Sons, Ltd.
711
Magn. Reson. Chem. 2014, 52, 711–714
Two types of samples were used in this study: calibration curve samples and spiked urine samples. In order to create calibration curves for MUS and IBO, 540 μl of the deionized H2O containing particular concentration of standard compound solution was mixed with 60 μl of phosphate buffer solution (PBS) containing 99.8% D2O, sodium azide, and the internal standard, deuterated 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid (TSP-d4). For spiked urine samples, freshly collected urine was split into 530 μl aliquots and placed in polypropylene Eppendorf tubes. Urine samples were mixed with 60 μl of PBS, the same as for calibration curves. Next, 10 μl of the standard compound solution was added to the sample (one at each time IBO or MUS), whereas in the control sample, it was replaced with 10 μl of the deionized H2O. All prepared samples containing standard solutions are listed in Table 1. The samples were mixed and centrifuged at 12 000 rpm for 5 min. In total, 550 μl supernatant was transferred into 5 mm NMR tubes.
S. Deja et al. Table 1. List of samples used and concentrations of standard compounds in calibration curve samples and spiked urine samples Muscimol (μg/ml) 277.78 166.67 111.11 55.56 27.78 13.89 8.33 5.56 2.78 1.39 55.56 38.89 27.78 22.22 16.67 5.56 2.78 1.94
Ibotenic acid (μg/ml) 416.67 250.00 166.67 83.33 41.67 20.83 12.50 8.33 4.17 2.08 83.33 58.33 41.67 33.33 25.00 8.33 4.17 2.92
Sample type Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Calibration curve Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine Spiked urine
acquisition time and quantification accuracy. Several mixing times were examined, namely 10, 100, 200, 400, 600, and 800 ms. The phase and baseline correction were performed manually. Regions on the spectra containing MUS and IBO at 5.8–5.9 ppm were utilized for signal integral calculations. Data preprocessing A Fourier transformation was performed on all free induction decays with 0.3 Hz line broadening. The phase and baseline corrections were manually conducted using Topspin 1.3 software (Bruker, GmBH, Germany). The spectra were calibrated to the TSP signal (δ = 0.0 ppm) and exported to MATLAB, where alignment of the region of interest (5.4–6.2 ppm) was performed using the icoshift algorithm.[12] The signals were integrated and expressed as raw metabolite integrals and as a ratio of raw metabolite integrals relative to the TSP signal. The calibration curves were obtained in MS Excel, where were calculated curves equations, standard deviation of the regression lines (SD), and limit of detection (LOD = 3.3(SD/slope of the curve)).
compounds, the exogenous substances that can be metabolized by humans and those that remain unchanged can be quickly identified provided they are concentrated enough.[15,16] The primary Amanita hallucinogenic compounds are IBO and MUS (Scheme 1), each of which has only two signals on the 1H NMR spectra. Aliphatic signals from the methine group and methylene protons were present; however, these signals may be obscured by urine metabolites, e.g. glucose, potential xenobiotics or residual H2O signal.[17,18] The only way to analyze both substances (IBO and MUS) was to examine resonance signals belonging to the isoxazole ring at 5.88 ppm and 5.86 (Fig. 1). Unfortunately, the compounds of interest only appeared over the urea signal, which limited accuracy and significantly lowered the detection limit. Therefore, the method to analyze the isoxazole proton on the 1D-NMR spectra among the urine matrix metabolites was developed. For this purpose, the 1D-NOESY spectra were used with a water presaturation pulse. The advantage of this pulse sequence is that protons exhibiting intermolecular exchange with water would be also attenuated during presaturation depending on the pulse sequence parameters.[15] To assess the detection limit, samples with various concentrations were measured, starting with the highest concentration and decreasing to 2–3 μg/ml. For each spectrum, various mixing times were used, which effectively suppressed the water signal and decreased the broad urea signal originating from exchangeable NH protons.[15] During mixing time optimization, the values were continuously increased from 10 to 800 ms. However, the optimal mixing time strongly depended on the urea content of the urine sample; at high signals, 600 ms was required, but at lower signals, 300–400 ms may be sufficient. Thus, 600 ms was used for all experiments.
Scheme 1. The structures of (a) ibotenic acid and (b) muscimol.
Results and discussion
712
Despite the many studies dedicated to the analysis of hallucinogenic compounds, which can be identified in biofluids after ingestion, there is a need for rapid and low-cost analytical methods to detect various compounds. This issue is especially important because of the increased popularity of the fungi and easy access to the natural not prohibited stimulants. The primary detection method is mass spectrometry, but special sample preparation procedures are needed, as well as the use of either GC or HPLC-MS tandem methods.[5,13] Because of the increasing number of available NMR spectrometers and their commercial use and hence of cost reducing of measurements, we have demonstrated the usefulness of an NMR method in the field. Approximately at least 20–40 compounds in urine of healthy donors can be characterized by NMR spectroscopy, where their analysis might be qualitative or quantitative.[14] Among these
wileyonlinelibrary.com/journal/mrc
Figure 1. Influence of increasing mixing time on the urea signal attenuation for (a) the muscimol sample (singlet at 5.86 ppm) and (b) the ibotenic acid sample (singlet at 5.88 ppm).
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 711–714
Determination of ibotenic acid and muscimol in urine
Figure 2. Effect of normalizing the signal integrity of hippuric acid, dimethylamine (DMA) and citric acid to the internal standard TSP: (a) raw spectrum and (b) spectrum after normalization.
When increasing the mixing time, some resonance signals experienced a considerable reduction of signal intensity. Some signals were not affected, e.g. hippuric acid, whereas others, such as citric acid, changed significantly (Fig. 2). The influence of mixing time on the resonance signal intensity of selected metabolites is given in Table 2. The relative standard deviation illustrates the average change among the measurements. The selected signals were normalized to TSP using the MATLAB program (Fig. 2). Because of the non-uniformity of the urine, the calibration curves (600 ms mixing time) were performed in PBS and showed linearity with R2 = 0.9996 for MUS over the range from 278 to 3 μg/ml (LOD = 13 μg/ml) and R2 = 0.9990 for IBO from 417 to 2 μg/ml (LOD = 30 μg/ml). The obtained curves with equations for MUS
y = 0.0031 × 0.0025 and IBO y = 0.0022 × 0.0044 were used to calculate the amount of MUS and IBO spiked in the urine (Fig. 3). The method showed very good precision for IBO even below LOD value down to 25.09 μg/ml (0.4 % difference), where the sample with 10 μg/ml showed a 20% deviation due to small signal-to-noise ratio (Table 3). The detection range was sufficient to cover the IBO range (32–55 μg/ml) in urine after fungi consumption.[13] However, the MUS level in human urine after fungi ingestion is much lower (10–7 μg/ml), and this method provided accuracy at 7 μg/ml with a 27% deviation, although this strongly depends on sample acquisition time, where the signal-to-noise ratio may be strongly improved.
Table 2. The influence of mixing times of 10–800 ms on the signal intensities of the selected metabolites
Table 3. The test sample concentrations of muscimol and ibotenic acid spiked in urine with % error from the calibration curve
Selected metabolites
1 2 3 4 5 6 7 8 9 10
Alanine Alanine/TSP Citric acid Citric acid/TSP Creatinine Creatinine/TSP Formic acid Formic acid/TSP Hippuric acid Hippuric acid/TSP
Magn. Reson. Chem. 2014, 52, 711–714
Muscimol
Relative standard deviation (%) 29.09 21.38 34.02 26.61 11.93 4.53 10.32 4.56 9.94 3.23
C (μg/ml) from NMR 58.26 41.81 28.81 23.68 18.68 7.06 4.84 3.48
Copyright © 2014 John Wiley & Sons, Ltd.
Ibotenic acid
% difference
C (μg/ml) from NMR
% difference
4.86 7.50 3.70 6.55 12.06 27.16 74.19 79.17
80.09 60.50 43.68 34.73 25.09 10.00 7.77 6.55
3.89 3.71 4.84 4.18 0.36 20.00 86.55 124.42
wileyonlinelibrary.com/journal/mrc
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No.
S. Deja et al.
Figure 3. Calculated calibration curves in PBS (black triangles) and overlapped spiked urine (red circles) for (a) muscimol and (b) ibotenic acid.
Conclusion In this study, a quick and inexpensive method was examined and used to analyze two common Amanita neurotoxins (MUS and IBO) spiked in urine samples. A 1D-NOESY experiment was performed on samples at various mixing times; a mixing time of 600 ms was adequate to decrease the overlapping urea signal and enabled the quantitative and qualitative analysis of the MUS and IBO compounds.
Acknowledgements The research was supported by the Wroclaw Research Centre EIT+ under the project named ‘Biotechnologies and advanced medical technologies’—BioMed (POIG.01.01.02-02-003/08) and financed by the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). Stanisław Deja was a recipient of PhD scholarship under a project funded by the European Social Fund.
References [1] D. Michelot, L. M. Melendez-Howell. Mycol. Res. 2003, 107, 131–146. doi:10.1017/S0953756203007305. [2] J. Ott, S. P. S. Wheaton, W. S. Chilton. Physiol. Chem. Phys. 1975, 7, 381–384. [3] R. J. Walker, G. N. Woodruff, G. A. Kerkut. Comp. Gen. Pharmacol. 1971, 2, 168–174.
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