QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Erratum All Versions of this Article: dmd.105.005835v1 34/2/281    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in DMD Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kamata, T. Articles by Tsuchihashi, H. Search for Related Content PubMed PubMed Citation Articles by Kamata, T. Articles by Tsuchihashi, H.

METABOLISM OF THE PSYCHOTOMIMETIC TRYPTAMINE DERIVATIVE 5-METHOXY-N,N-DIISOPROPYLTRYPTAMINE IN HUMANS: IDENTIFICATION AND QUANTIFICATION OF ITS URINARY METABOLITES

Forensic Science Laboratory, Osaka Prefectural Police Headquarters, Osaka, Japan (T.K., M.K., H.T.K., A.M., N.S., K.Z., M.N., H.T.); and Department of Legal Medicine, University of Tsukuba, Tsukuba, Japan (E.T., K.H.)

(Received June 15, 2005; accepted November 7, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

View larger version (6K): [in this window] [in a new window]   FIG. 1. Chemical structure of 5-MeO-DIPT.

Although research on the metabolism of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) in mammals (Sitaram et al., 1987) provides some useful information for analyzing 5-MeO-DIPT metabolites in human body fluids, only a few data have been reported for its metabolism in humans. Recently, "tentative identification" of several metabolites in humans, but without using authentic standards, was reported (Meatherall and Sharma, 2003; Wilson et al., 2005).

In this report we have aimed for the careful investigation and indisputable identification of the metabolites of 5-MeO-DIPT in humans. The authors for the first time synthesized the authentic standards of its metabolites that were predicted based on previous studies (Meatherall and Sharma, 2003; Wilson et al., 2005; Tsutsumi et al., 2005a,b). Utilizing the authentic standards, several urine specimens from 5-MeO-DIPT users were analyzed by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The excretion profiles are presented, and the metabolic pathways of 5-MeO-DIPT are discussed.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. 5-MeO-DIPT hydrochloride, 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT) hydrochloride, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) hydrochloride, 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), and 5-methoxy-N,N-diisopropyltryptamine-N-oxide (5-MeO-DIPT-NO) were synthesized in our laboratory according to previously published methods with partial modification, as detailed below (their chemical structures can be seen in Fig. 5). Every synthesized compound was ensured to be >95% pure based on high-resolution MS analysis by the flow-injection method. Stock standard solutions of these five compounds were prepared in methanol (1 mg/ml each) and then diluted to appropriate concentrations with distilled water or control human urine, immediately before use. The internal standard (I.S.) 5-methyltryptamine (5-MT) hydrochloride was purchased from Aldrich Chemical Co. (Milwaukee, WI), and an I.S. solution (200 ng/ml) was prepared in distilled water. Acetonitrile and methanol were of high-performance liquid chromatography grade, and other chemicals used were of analytical grade. Sulfatase/ß-glucuronidase (Helix pomatia, type H-1) was obtained from Sigma-Aldrich (St. Louis, MO). N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) used for trimethylsilyl (TMS) derivatization was purchased from Wako Pure Chemicals (Osaka, Japan).

View larger version (21K): [in this window] [in a new window]   FIG. 5. Metabolic pathways of 5-MeO-DIPT in humans revealed in this study. 5-MeO-DIPT is mainly metabolized by demethylation and hydroxylation. Most of the 6-OH-5-MeO-DIPT formed by hydroxylation is excreted as its conjugates.

5-MeO-NIPT (II). II was synthesized according to the above-mentioned procedure, with a slight modification: instead of excess 2-iodopropane, an equivalent amount of 2-iodopropane was added to 5-methoxytryptamine, and the resultant reaction mixture was purified on a column packed with Chromatorex NH, using chloroform as an eluent (Fuji Silysia Chemical, Ltd., Kasugai, Japan).

5-OH-DIPT (III). III was synthesized in analogy to the modified procedure for 5-hydroxy-N,N-dimethyltryptamine (Shulgin and Shulgin, 1997b): diisopropylamine was used instead of dimethylamine.

6-OH-5-MeO-DIPT (IV). IV was synthesized according to the procedures of Shulgin and Shulgin (1997c,d), with modifications as follows. To concentrated nitric acid, which was stirred and cooled in an external ice-bath, finely powdered 4-benzyloxy-3-methoxybenzaldehyde (Avocado Research Chemicals, Ltd., Heysham, UK) was added. (The temperature must not be allowed to rise above 0°C.) After 2 h of additional stirring, the reaction mixture was poured over chipped ice, and the product was extracted with ethyl acetate. The extract was washed with a saturated aqueous sodium bicarbonate solution and water to remove remaining acid. After evaporation, the residue was subjected to column chromatography, using a silica gel column and an ethyl acetate/n-hexane mixture (1:1 v/v) as a developing solvent, to obtain 2-nitro-4-benzyloxy-3-methoxybenzaldehyde. (The yield in this step was 70%.)

A solution of 2-nitro-4-benzyloxy-3-methoxybenzaldehyde in glacial acetic acid was treated with nitromethane (Wako Pure Chemicals), followed with anhydrous ammonium acetate. After being held at reflux for 2 h, the reaction mixture was poured into ice water. The product was extracted with ethyl acetate, and the extract was evaporated to dryness after washing with water. The residue was subjected to column chromatography, using a silica gel column and an ethyl acetate/n-hexane mixture (1:2 v/v) as a developing solvent, to isolate 2,2'-dinitro-4-benzyloxy-3-methoxystyrene (78%).

To glacial acetic acid, 2,2'-dinitro-4-benzyloxy-3-methoxystyrene and electrolytic powdered iron were added, stirred, and heated gently until an exothermic reaction set in, and this was maintained at a controlled pace with external cooling. When the spontaneous reaction had subsided, the reaction mixture was refluxed for 15 min, cooled, neutralized with sodium hydroxide, and extracted with ethyl acetate. The extract was evaporated. The residue was subjected to column chromatography, using a Chromatorex NH column and an ethyl acetate/n-hexane mixture (1:3, v/v) as a developing solvent, to obtain 6-benzyloxy-5-methoxyindole (30%).

To a well stirred, cold solution of 6-benzyloxy-5-methoxyindole in anhydrous diethyl ether, a solution of oxalyl chloride (Wako Pure Chemicals) in diethyl ether was added dropwise with protection from atmospheric moisture. The reaction mixture was stirred for an additional 20 min, and the intermediate indoleglyoxyl chloride was separated as a crystalline solid but was not isolated. This was treated with 40% solution of diisopropyl amine in anhydrous diethyl ether, dropwise, until the pH reached 8 to 9. The reaction mixture was diluted with chloroform and shaken with 5% aqueous potassium bisulfate (Wako Pure Chemicals) solution, followed by a saturated aqueous sodium bicarbonate solution. After drying, the organic solvent was removed. The residue was subjected to column chromatography, using a Chromatorex NH column and an ethyl acetate/n-hexane mixture (1:5 v/v) as a developing solvent, to isolate 6-benzyloxy-5-methoxyindol-3-yl-N,N-diisopropylglyoxylamide (55% in these two steps).

To a well stirred suspension of lithium aluminum hydride in dry tetrahydrofuran (THF), a solution of 6-benzyloxy-5-methoxyindol-3-yl-N,N-diisopropylglyoxylamide in anhydrous THF was added dropwise. The mixture was brought to a reflux temperature, held there for 15 min, and allowed to return to room temperature. The excess hydride was destroyed by adding ethyl acetate and water. The solids were removed by filtration, the filter cake was washed with THF, and the pooled filtrate and washings were evaporated under vacuum. The residue was subjected to column chromatography, using a Chromatorex NH column and an ethyl acetate/n-hexane mixture (1:5, v/v) as a developing solvent, to obtain 6-benzyloxy-5-methoxy-N,N-diisopropyltryptamine (35%).

A solution of 6-benzyloxy-5-methoxy-N,N-diisopropyltryptamine in methanol containing 10% palladium-activated carbon catalyst was shaken under 3 atm of hydrogen for 6 h, and the solids were removed by filtration. Evaporation of the solvent under vacuum gave a residue. The residue was subjected to column chromatography using a Chromatorex NH column and an ethyl acetate/n-hexane mixture (1:1 v/v) as a developing solvent, to isolate 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (88%). The overall isolated yield was 2.8%.

5-MeO-DIPT-NO (V). V was synthesized from I, according to the procedure of Cymerman Craig and Purushothaman (1970).

The identities of all synthesized compounds were confirmed by high-resolution MS and MS/MS in the flow-injection method, in addition to GC/electrospray ionization (EI) MS with and without TMS derivatization and LC/ESI MS. The GC/EI MS and LC/ESI MS data are described under Results. The high-resolution mass spectral data (calculated exact mass is given in parentheses) are as follows: I: m/z 275.2102 [M + H]⁺ (275.2133), 174.0896 [M – (C₃H₇)₂N]⁺ (174.0919), 114.1314 (C₃H₇)₂NCH₂⁺ (114.1283). II: m/z 233.1659 [M + H]⁺ (233.1654), 174.0894 [M – C₃H₇NH]⁺ (174.0919). III: m/z 261.1967 [M + H]⁺ (261.1967), 160.0756 [M – (C₃H₇)₂N]⁺ (160.0762), 114.1312 (C₃H₇)₂NCH₂⁺ (114.1283). IV: m/z 291.2079 [M + H]⁺ (291.2072), 190.0855 [M – (C₃H₇)₂N]⁺ (190.0852). V: m/z 291.2099 [M + H]⁺ (291.2072), 174.0897 [M – (C₃H₂)₂NO]⁺ (174.0919).

Apparatus. GC/MS was carried out on a GCMS QP-2010 (Shimadzu, Kyoto, Japan). A fused-silica capillary column, DB-5MS (30 m x 0.25 mm i.d.; 0.25 µm; J&W Scientific, Rancho Cordova, CA) was used for separation. Injections were effected automatically in the splitless mode at 260°C. The column oven temperature was maintained at 80°C for 2 min and then raised at 20°C/min to 310°C. The transfer line temperature was set at 260°C. High-purity helium, at a flow rate of 3 ml/min, was used as the carrier gas. The EI operating parameters were as follows: source temperature, 200°C; electron energy, 70 eV; ion multiplier gain, 1.2 kV. Data were collected from m/z 40 to 600 at a scan rate of 0.5 s/scan.

LC/MS was performed on a ZMD system equipped with an Alliance 2690 pump and an ESI interface (Waters, Milford, MA). The capillary and cone voltages were set at 2.0 kV and 25 V, respectively. The other ESI operating parameters were as follows: ion-source temperature, 100°C; desolvation temperature, 300°C; nitrogen gas, 450 l/h; and multiplier voltage, 650 V. Under these conditions, full-scan data were acquired from m/z 100 to 600 in the centroid mode, using a cycle time of 1.0 s and an interscan time of 0.1 s. The quantitative measurements were accomplished in the selected ion monitoring mode by monitoring each protonated molecule as follows: m/z 275 for 5-MeO-DIPT, m/z 233 for 5-MeO-NIPT, m/z 261 for 5-OH-DIPT, m/z 291 for 6-OH-5-MeO-DIPT, and m/z 175 for 5-MT, by an internal standard method. The chromatographic separation was carried out on a semi-micro L-column ODS column (1.5 mm i.d.x150 mm; Chemicals Evaluation and Research Institute, Tokyo, Japan) with a binary mobile phase of methanol and 10 mM ammonium formate (pH 3.5), using a linear gradient (methanol, 25– 40% in 5 min). The flow rate was set at 0.1 ml/min, and the entire flow was introduced into the ESI source.

LC-MS/MS was performed on a Quattro LC (Waters) system equipped with an Agilent 1100 pump (Agilent Technologies, Palo Alto, CA) and an ESI interface under the same chromatographic conditions and operating parameters, except that the ion-source temperature was set at 280°C. Argon was used as the collision gas at collision energies of 15 eV (for conjugates of 6-OH-5-MeO-DIPT) and 20 eV (for conjugates of 5-OH-DIPT). Ions of m/z 341, 437, 371, and 467, corresponding to the protonated molecules of 5-OH-DIPT-sulfate, 5-OH-DIPT-glucuronide, 6-OH-5-MeO-DIPT-sulfate, and 6-OH-5-MeO-DIPT-glucuronide, respectively, were selected as precursor ions.

High-resolution MS and MS/MS were performed on an LCMS-IT-TOF (Shimadzu). The probe voltage was +4.5 kV, and both the curved desolvation line temperature and the block heater temperature were 200°C. The ion accumulation time and isolation time were set at 30 ms and 20 ms, respectively. Argon was used as collision gas at 50% of introduction. The collision-induced dissociation energy and the collision-induced dissociation time were 50% and 30 ms, respectively.

Specimen Collection. Urine specimens were voluntarily provided from two 5-MeO-DIPT users. User A (24-year-old Japanese male) consumed a capsule containing approximately 50 mg of 5-MeO-DIPT hydrochloride through the anus (exact dosage unclear). About 30 min later, he began intensive sweating, became hyperaggressive, and was taken to the emergency department with dementia. His urine specimens were collected at approximately 11 h and 35 h post-intake. User B (22-year-old Japanese male) drank fruit juice spiked with 5-MeO-DIPT hydrochloride (dosage unclear). He was brought to the hospital with strong dementia. The urine specimen was collected at approximately 18 h post-intake. Urine specimens were immediately frozen and stored at –20°C until analysis.

Sample Preparation. Extraction and TMS Derivatization for GC/MS. A 1-ml sample of a urine specimen was adjusted to pH 8 with 2.8% ammonium hydroxide, mixed vigorously with 500 µl of a chloroform-isopropyl alcohol mixture (3:1 v/v), and centrifuged at 1500g for 10 min. The organic layer was recovered and dehydrated with anhydrous sodium sulfate. A 1-µl aliquot was injected into the GC/MS system. A 100-µl aliquot of the extract was transferred into a screw-capped glass tube and was derivatized by adding 100 µl of MSTFA and then heating at 60°C for 30 min. A 1-µl aliquot of the reaction mixture was manually injected into the GC/MS system.

Enzymatic Hydrolysis. To a 300-µl urine specimen was added 30 µl of 0.1 M ascorbic acid, and the specimen was adjusted to pH 5 with 10% acetic acid. After adding 60 µl of 0.5 M acetate buffer (pH 5.0) and H. pomatia sulfatase/ß-glucuronidase (300 and 8680 Fishman units, respectively), the specimen was incubated at 37°C for 2 h to hydrolyze conjugates.

Extraction for LC/MS of Free-Form Metabolites. Urine specimens before and after hydrolysis were adjusted to pH 8 with 2.8% ammonium hydroxide and mixed vigorously with 2 volumes of a chloroform-isopropyl alcohol mixture (3:1 v/v). After centrifugation, the organic layers were recovered. The extraction was repeated twice and the organic extracts were combined, and this combined extract was evaporated to dryness under a gentle stream of nitrogen at 40°C. The residue was dissolved in 100 µl of distilled water, and the I.S. solution was added. This was then filtered through a 0.2-µm membrane filter, and an aliquot of 5 µl was automatically injected into the LC/MS system.

Extraction for LC/MS and LC-MS/MS of Conjugates. Urine specimens before and after hydrolysis were each mixed vigorously with 2 volumes of methanol. After centrifugation, the supernatant was recovered and evaporated to dryness. The residue was dissolved in 100 µl of distilled water and filtered. Aliquots of 5 µl were automatically injected into the LC/MS and LC-MS/MS systems.

Validation of the LC/MS Procedure. To quantify 5-MeO-DIPT and its metabolites in urine, the LC/MS procedure optimized was validated. A 300-µl drug-free urine spiked with the synthesized standards at 1 µg/ml each was processed as described above. The residue was dissolved in 100 µl of distilled water and added to 100 µl of 200 ng/ml I.S. (5-MT) solution. A 5-µl aliquot was injected into the LC/MS system in the selected ion monitoring mode, where the protonated molecules of each analyte were selected as the monitoring ions, and the peak area ratios to I.S. were calculated. The recoveries at 1 µg/ml were 93.1%, 92.3%, 95.7%, and 105% for 5-MeO-DIPT, 5-MeO-NIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT, respectively (n = 5). The detection limits were 0.03 µg/ml for 6-OH-5-MeO-DIPT and 0.003 µg/ml for the others. Calibration curves constructed by the I.S. method showed good linearities over the ranges from 0.1 to 10 µg/ml for 6-OH-5-MeO-DIPT and from 0.01 to 10 µg/ml for the others. The within-day relative standard deviations (evaluated at 1 µg/ml, n = 5) ranged from 3.35 to 5.17% for all of the analytes. These results guaranteed the reliability of the present procedure for the analysis of urine specimens from 5-MeO-DIPT users.

	Results

Top Abstract Materials and Methods Results Discussion References

 
GC/MS and LC/MS of Predicted Metabolite Standards. The EI mass spectra of the authentic standards, except for 5-MeO-DIPT-NO, indicated their characteristic structures: predominant ions due to the -cleavage of amine moieties (m/z 72 for 5-MeO-NIPT; m/z 114 for 5-MeO-DIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT) and some other ions, including a very small molecular ion (m/z 274 for 5-MeO-DIPT, m/z 232 for 5-MeO-NIPT, m/z 260 for 5-OH-DIPT, and m/z 290 for 6-OH-5-MeO-DIPT) (data not shown). 5-MeO-DIPT-NO was not detectable by GC/MS because N-oxide metabolites readily degrade in the injection port of the gas chromatograph.

GC/MS was also carried out after TMS derivatization because of its higher sensitivity and clearer identification of amines and phenols. The EI mass spectra of their TMS derivatives also had a molecular ion (m/z 346 for 5-MeO-DIPT-TMS, m/z 304 for 5-MeO-NIPT-TMS, m/z 404 for 5-OH-DIPT-di-TMS, and m/z 434 for 6-OH-5-MeO-DIPT-di-TMS), but their relative intensities were still very low, as in the case without derivatization (data not shown).

Unlike GC/MS spectra, the ESI mass spectra of the authentic standards taken by LC/MS (data not shown) were characterized by the predominant protonated molecules at m/z 275 for 5-MeO-DIPT, m/z 233 for 5-MeO-NIPT, m/z 261 for 5-OH-DIPT, m/z 291 for 6-OH-5-MeO-DIPT, and 5-MeO-DIPT-NO. It should be noted that the ESI mass spectrum of 5-MeO-DIPT-NO had a weak dimerization ion [2M + H]⁺, which often appears specifically for amine N-oxides, at m/z 581.

Identification of the Metabolites in Urine. The extracts of the unhydrolyzed urine specimens were first analyzed by GC/MS with and without TMS derivatization, and the retention times and mass spectra of compounds detected were compared with those of the standards. As a result, free-form 5-MeO-DIPT, 5-MeO-NIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT were detected in the extracts without derivatization, and TMS derivatives of these four compounds were also clearly detected in the derivatized extracts.

LC/ESI MS was next carried out under the optimized conditions, and the results were compared with those of the authentic standards. Figure 2 shows the extracted ion chromatograms obtained from User A's urine (11 h post-intake). Although 5-MeO-DIPT, 5-MeO-NIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT were confirmed, no trace of 5-MeO-DIPT-NO was detected in this specimen.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Extracted ion chromatograms obtained from a 5-MeO-DIPT user's urine specimen by LC/MS.

To examine the stability of the predicted N-oxide metabolite, six control urine samples from drug-free volunteers were spiked with 5-MeO-DIPT-NO at 1 µg/ml, and the samples were stored at –20°C for 1 month. No noticeable decrease of 5-MeO-DIPT-NO was detected in any of the samples tested. Thus, the absence of 5-MeO-DIPT-NO was not attributed to its denaturation or decomposition.

Excretion of 5-MeO-DIPT and Its Metabolites. The concentrations of 5-MeO-DIPT and its three metabolites identified in the urine specimens were quantified by the validated LC/MS procedure, using the calibration curves constructed, and the excretion profiles were investigated. Also, the concentrations before and after enzymatic hydrolysis were compared. The results are summarized in Table 1. Although 5-MeO-DIPT, 5-MeO-NIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT were detected in all of the samples, no trace of 5-MeO-DIPT-NO was detected in any of the specimens examined.

Because excretion of the glucuronides and sulfates was expected for 5-OH-DIPT and 6-OH-5-MeO-DIPT from their increased levels after hydrolysis, their direct detection was attempted by LC-MS/MS. Typical extracted ion chromatograms, obtained before and after sulfatase/ß-glucuronidase treatment, are shown in Fig. 3. Unhydrolyzed urine gave four distinctive peaks at retention times of 4.3, 3.0, 4.1, and 4.8 min on the extracted ion chromatograms at m/z 341, 437, 371, and 467, respectively, which correspond to the protonated molecules of 5-OH-DIPT-sulfate, 5-OH-DIPT-glucuronide, 6-OH-5-MeO-DIPT-sulfate, and 6-OH-5-MeO-DIPT-glucuronide, respectively. The sulfatase/ß-glucuronidase treatment for 2 h resulted in decreases in the peak areas at 4.3 and 4.1 min, and disappearance of the peaks at 3.0 and 4.8 min, whereas the peak areas of 5-OH-DIPT and 6-OH-5-MeO-DIPT significantly increased. To confirm these peak assignments, these peaks were further analyzed by MS/MS. The results are summarized in Fig. 4. The results indicate that the metabolites eluting at 4.3 and 3.0 min were 5-OH-DIPT-sulfate and 5-OH-DIPT-glucuronide, as indicated by the neutral loss of 80 (the sulfate group), and that of 176 (the glucuronyl group), to produce the protonated molecule of 5-OH-DIPT (m/z 261) and its characteristic substructural ions (m/z 114 and 160). In the same manner, the metabolites eluting at 4.1 and 4.8 min were proved to be 6-OH-5-MeO-DIPT-sulfate and 6-OH-5-MeO-DIPT-glucuronide; the neutral losses of 80 and 176 resulted in the protonated molecule of 6-OH-5-MeO-DIPT (m/z 291) and its characteristic substructural ion (m/z 114).

View larger version (32K): [in this window] [in a new window]   FIG. 3. Comparison of LC/MS-extracted ion chromatograms obtained from a 5-MeO-DIPT user's urine specimen before (left) and after (right) enzymatic hydrolysis. The urine specimen was hydrolyzed by incubation at 37°C for 2 h with H. pomatia sulfatase/ß-glucuronidase (300 and 8680 Fishman units, respectively).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Observed fragment ions on LC-MS/MS of four conjugates in a 5-MeO-DIPT user's urine specimen. Collision energies were set at 15 eV (for conjugates of 6-OH-5-MeO-DIPT) and 20 eV (for conjugates of 5-OH-DIPT). Ions corresponding to each protonated molecule of conjugates were selected as precursor ions.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Previous studies on the rat reported metabolic routes of DMT and 5-MeO-DMT, which include N-oxidation, N-demethylation, O-demethylation, and oxidative deamination (Sitaram et al., 1987). Based on that previous report, 5-MeO-NIPT, 5-OH-DIPT, and 5-MeO-DIPT-NO were predicted as the major urinary metabolites of 5-MeO-DIPT. In addition, Morano et al. (1993) proposed that etryptamine (ethyltryptamine) is metabolized mainly by 6-hydroxylation, like other indole derivatives, which suggested the possibility of hydroxylation at the 6-position of 5-MeO-DIPT into 6-OH-5-MeO-DIPT. Thus, the present authors selected 6-OH-5-MeO-DIPT, in addition to the above-mentioned three metabolites, as the expected major metabolites that retain the structural characteristics of the parent drug 5-MeO-DIPT. Based on the GC/MS and LC/MS analyses in this study, 5-MeO-DIPT, 5-MeO-NIPT, 5-OH-DIPT, and 6-OH-5-MeO-DIPT were indisputably identified in human urine from 5-MeO-DIPT users.

In this study, no trace of 5-MeO-DIPT-NO was detected in any of the urine specimens from 5-MeO-DIPT users. In the previous study by Sitaram et al. (1987) on the metabolism of 5-MeO-DMT, an analog of 5-MeO-DIPT, 5-MeO-DMT-N-oxide was identified as the abundant and characteristic urinary metabolite in the rat. This difference is probably attributed to steric interference with N-oxidation on the tertiary nitrogen atom by the two bulky isopropyl moieties, although the possibility of interspecies variations should not be excluded. Also, no noticeable decrease of 5-MeO-DIPT-NO during sample storage was detected, as mentioned above. Thus, there is probably no metabolic transformation of 5-MeO-DIPT into 5-MeO-DIPT-NO in humans.

Our studies confirm that 5-MeO-NIPT and 5-OH-DIPT are metabolites found in human urine as predicted by Meatherall and Sharma (2003) and Wilson et al. (2005). However, 6-OH-5-MeO-DIPT was also identified, rather than 5-MeO-DIPT-N'-oxide, as the GC/MS product close to the 5-OH-DIPT peak. Because 5-MeO-DIPT-N'-oxide and 6-OH-5-MeO-DIPT have the same molecular weight, we concluded that the metabolite that Meatherall's team expected to be 5-MeO-DIPT-N'-oxide was probably the 6-OH-5-MeO-DIPT identified here.

Excretion of 5-MeO-DIPT and Its Metabolites. As summarized in Table 1, enzymatic hydrolysis increased 6-OH-5-MeO-DIPT concentration to almost the same level as that of 5-OH-DIPT, although no notable increase was observed for 5-OH-DIPT. These facts indicate that 5-OH-DIPT and 6-OH-5-MeO-DIPT are abundant and characteristic metabolites and that N-deisopropylation is a minor metabolic pathway for 5-MeO-DIPT.

Based on the quantitative results listed in Table 1, it should be noted that the conjugation rate of 5-OH-DIPT seemed to be very low, although most of the 6-OH-5-MeO-DIPT was excreted as its conjugates. For determining exact rates of sulfates and glucuronides, analysis should be done using authentic standards of conjugates, or by performing selective hydrolysis using sulfatase and ß-glucuronidase inhibitors. However, in the direct analysis of the conjugates of hydroxylated metabolites of phenethylamine-type drugs by LC/ESI MS and LC/ESI MS/MS, we found that the sensitivities in detecting glucuronides were 1.2 to 2.3 times higher than those of sulfates in the LC/ESI MS of p-hydroxymethamphetamine and 4-hydroxy-3-methoxymethamphetamine, the major metabolites of methamphetamine and 3,4-methylenedioxymethamphetamine. Based on these facts, the present data in Fig. 3 suggest that 5-OH-DIPT is excreted in urine mainly in the unconjugated form, whereas 6-OH-5-MeO-DIPT is excreted mostly as its sulfate.

These results revealed three metabolic pathways of considerable quantitative significance for 5-MeO-DIPT in humans. As shown in Fig. 5, the first pathway leads, via side chain degradation by O-demethylation, to 5-OH-DIPT, partly followed by conjugation to form its sulfate and glucuronide. The second pathway leads, via direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5, after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-OH-5-MeO-DIPT, followed by conjugation to its sulfate and glucuronide. Although the hydroxylated 5-OH-DIPT (Fig. 5) is also expected to be methylated to form 5-hydroxy-6-methoxy-diisopropyltryptamine, the positional isomer of 6-OH-5-MeO-DIPT, 6-OH-5-MeO-DIPT and 5-hydroxy-6-methoxy-diisopropyltryptamine should be distinguished by their retention times on GC/MS and LC/MS according to our previous studies. The positional isomers of N-benzylpiperazine metabolites (Tsutsumi et al., 2005b) and methylenedioxymethamphetamine (Ecstasy) metabolites (unpublished data) had been well separated by GC/MS and LC/MS, and the urinary hydroxy-methoxy-diisopropyltryptamine detected in this study completely agreed with 6-OH-5-MeO-DIPT. We, therefore, concluded that the hydroxylated metabolite is 6-OH-5-MeO-DIPT. The third pathway leads, via side chain degradation by N-deisopropylation, to the corresponding secondary amine, 5-MeO-NIPT. Of these, 5-OH-DIPT and 6-OH-5-MeO-DIPT were of the major metabolites. However, further investigation of hydrolysis conditions is necessary to exactly evaluate the contributions of demethylation and hydroxylation to 5-MeO-DIPT metabolism, because the hydrolysis of 5-OH-DIPT-sulfate and 6-OH-5-MeO-DIPT-sulfate could not be completed under the current conditions (Fig. 3). Also, a more quantitative experiment using radiolabeled 5-MeO-DIPT in an animal model might be very useful for more detailed survey of the metabolism of 5-MeO-DIPT. It is well known that oxidative N-deamination is a major metabolic pathway for 5-MeO-DMT in the rat (Sitaram et al., 1987) and, thus, it is expected to be an important metabolic pathway for 5-MeO-DIPT in humans. However, possible metabolites like 5-methoxyindoleacetic acid and 5-hydroxyindoleacetic acid, formed by the oxidative deamination, retain few structural characteristics of the parent compound. In fact, 5-hydroxyindoleacetic acid, which is commercially available, is known to be present at low µg/ml levels in the urine of healthy humans. Thus, the identification of characteristic urinary metabolites of 5-MeO-DIPT reported here will be of importance in forensic and clinical urine analysis. In addition, the present study will provide useful information in the urine analysis for unknown tryptamine-type drugs that may be encountered in the future.

	Footnotes

 
This study was supported by a grant-in-aid from Japan Society for the Promotion of Science (No. 16915019).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.005835.

ABBREVIATIONS: 5-MeO-DIPT, 5-methoxy-N,N-diisopropyltryptamine; DMT, dimethyltryptamine; EI, electron ionization; ESI, electrospray ionization; GC, gas chromatography; I.S., internal standard; LC, liquid chromatography; 5-MeO-DMT, 5-methoxy-N,N-dimethyltryptamine; 5-MeO-DIPT-NO, 5-methoxy-N,N-diisopropyltryptamine-N-oxide; 5-MeO-NIPT, 5-methoxy-N-isopropyltryptamine; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MSTFA, N-methyl-N-trimethylsilyltrifluoroacetamide; 5-MT, 5-methyltryptamine; 5-OH-DIPT, 5-hydroxy-N,N-diisopropyltryptamine; 6-OH-5-MeO-DIPT, 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine; THF, tetrahydrofuran; TMS, trimethylsilyl.

Address correspondence to: Tooru Kamata, Forensic Science Laboratory, Osaka Prefectural Police Headquarters, 1-3-18, Hommachi, Chuo-ku, Osaka 541-0053, Japan. E-mail: t-kamata{at}mahoroba.ne.jp

	References

Top Abstract Materials and Methods Results Discussion References

 


Cymerman Craig J and Purushothaman KK (1970) An improved preparation of tertiary amine N-oxides. J Org Chem 35: 1721–1722.[CrossRef][Medline]

[DEA] Drug Enforcement Administration (DEA), Department of Justice (2001) An encounter with 5-methoxy-N,N-diisopropyltryptamine. Microgram Bull 34: 126.

[DEA] Drug Enforcement Administration (DEA), Department of Justice (2003) Schedules of controlled substances: temporary placement of alpha-methyltryptamine and 5-methoxy-N,N-diisopropyltryptamine into Schedule I. Final rule. Fed Regist 68: 16427–164230.[Medline]

Katagi M, Tatsuno M, Miki A, Nishikawa M, Nakajima K, and Tsuchihashi H (2001) Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Biomed Sci 759: 125–133.[CrossRef]

Katagi M, Tatsuno M, Miki A, Nishikawa M, and Tsuchihashi H (2000) Discrimination of dimethylamphetamine and methamphetamine use: simultaneous determination of dimethylamphetamine-N-oxide and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Anal Toxicol 24: 354–358.[Medline]

Katagi M, Tsutsumi H, Miki A, Nakajima K, and Tsuchihashi H (2002) Analysis of clandestine tablets of amphetamines and their related designer drugs encountered in recent Japan. Jpn J Forensic Toxicol 20: 303–319.

Meatherall R and Sharma P (2003) Foxy, a designer tryptamine hallucinogen. J Anal Toxicol 27: 313–317.[Medline]

Morano RA, Spies C, Walker FB, and Plank SM (1993) Fatal intoxication involving etryptamine. J Forensic Sci 38: 721–725.[Medline]

Shulgin AT and Carter MF (1980) N,N-Diisopropyltryptamine (DIPT) and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT). Two orally active tryptamine analogs with CNS activity. Commun Psychopharmacol 4: 363–369.[Medline]

Shulgin A and Shulgin A (1997a) TiHKAL: The Continuation, pp 527–531. Transform Press, Berkeley, CA.

Shulgin A and Shulgin A (1997b) TiHKAL: The Continuation, pp 473–479. Transform Press, Berkeley, CA.

Shulgin A and Shulgin A (1997c) TiHKAL: The Continuation, pp 503–504. Transform Press, Berkeley, CA.

Shulgin A and Shulgin A (1997d) TiHKAL: The Continuation, pp 508–512. Transform Press, Berkeley, CA.

Sitaram BR, Lockett L, Talomsin R, Blackman GL, and McLeod WR (1987) In vivo metabolism of 5-methoxy-N,N-dimethyltryptamine and N,N-dimethyltryptamine in the rat. Biochem Pharmacol 36: 1509–1512.[Medline]

Tsutsumi H, Katagi M, Miki A, Shima N, Kamata T, Nakajima K, Inoue H, Kishi T, and Tsuchihashi H (2005a) Isolation, identification and excretion profile of the principal urinary metabolite of the recently banned designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rats. Xenobiotica 35: 107–116.[Medline]

Tsutsumi H, Katagi M, Miki A, Shima N, Kamata T, Nishikawa M, Nakajima K, and Tsuchihashi H (2005b) Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine. J Chromatogr B Biomed Sci 819: 315–322.

Wilson JM, McGeorge F, Smolinske S, and Meatherall R (2005) A foxy intoxication. Forensic Sci Int 148: 31–36.[Medline]

Related articles in DMD:

CORRECTION TO "METABOLISM OF THE PSYCHOTOMIMETIC TRYPTAMINE DERIVATIVE 5-METHOXY-N,N-DIISOPROPYLTRYPTAMINE IN HUMANS: IDENTIFICATION AND QUANTIFICATION OF ITS URINARY METABOLITES"

DMD 2006 34: 512. [Full Text]  

This Article Abstract Full Text (PDF) Erratum All Versions of this Article: dmd.105.005835v1 34/2/281    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in DMD Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kamata, T. Articles by Tsuchihashi, H. Search for Related Content PubMed PubMed Citation Articles by Kamata, T. Articles by Tsuchihashi, H.