Metabolite of (-)-trans-delta 8-tetrahydrocannabinol: identification and synthesis.

The major metabolite of (-)-trans-Delta(8)-tetrahydrocannabinol observed in vivo and formed by hepatic microsomes in vitro is 11-hydroxy-trans-Delta(8)-tetrahydrocannabinol. The metabolite was identified spectroscopically and was synthesized from trans-Delta(8)-tetrahydrocannabinol. In tests with rats, the metabolite produced behavioral effects similar to those imparted by Delta(8)- and Delta(9)-tetrahydrocannabinol.

Quantitative determination of endogenous sorbitol and fructose in human erythrocytes by atmospheric-pressure chemical ionization LC tandem mass spectrometry.

Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0-10,000 ng/mL (correlation coefficient >0.999) for sorbitol-13C6 and 250-50000 ng/mL (correlation coefficient >0.999) for fructose-13C6 in human RBCs.

Disposition of intravenous pirmenol.

Twelve patients having frequent premature ventricular complexes (PVCs) averaging more than 60 per hour received a single 150-mg intravenous dose of pirmenol. Plasma pirmenol concentration declined biexponentially following the infusion and was analyzed according to a two-compartment open model. Following an erratic distribution phase, the terminal elimination half-life ranged from 4.2 to 16.9 hours, with a geometric mean of 7.6 hours. Total body clearance averaged 164 +/- 58 ml/min, and the mean volume of distribution was 1.45 +/- 0.38 liter/kg. Renal clearance averaged 46.6 +/- 21.2 ml/min, representing 30 +/- 10 per cent of total body clearance. Excretion of unchanged drug in the urine averaged 31.8 +/- 8 per cent of the dose. Renal clearance and elimination half-life were correlated (r = -0.61, P less than 0.05). Eight of the 12 patients achieved greater than 95 per cent suppression of PVCs with a duration between 20 minutes and 23 hours. These favorable pharmacokinetics indicate that pirmenol may be a useful addition to the therapy of ventricular arrhythmias.

Naltrexone in autistic children: an acute open dose range tolerance trial.

The safety and efficacy of naltrexone was explored in an open acute dose range tolerance trial in 10 hospitalized autistic children, ages 3.42 to 6.50 years (mean, 5.04). Naltrexone was given in ascending doses: 0.5, 1.0, and 2.0 mg/kg/day. Behavioral side effects were observed as early as 1/2 hour after dosing. Ratings on the Children's Psychiatric Rating Scale showed that withdrawal was reduced across all three dose levels; administration of 0.5 mg/kg/day dose resulted in increased verbal production; and the 2.0 mg/kg/day dose resulted in reduction of sterotypies. Mild sedation of brief duration was the only side effect. Electrocardiogram, liver function tests, and all other laboratory studies remained unchanged throughout the study. These preliminary findings require replication in a larger sample of patients under double-blind and placebo controlled condition.

Long-term alteration in the central monoaminergic systems of the rat by 2,4,5-trihydroxyamphetamine but not by 2-hydroxy-4,5-methylenedioxymethamphetamine or 2-hydroxy-4,5-methylenedioxyamphetamine.

The long-term effects of three metabolites of 3,4-methylenedioxymethamphetamine (MDMA) on the central monoaminergic systems of the rat were examined. Seven days after the intracerebroventricular administration of 0.25 and 0.5 mumol 2,4,5-trihydroxyamphetamine, hippocampal tryptophan hydroxylase (TPH) activity was reduced to 5 and 1% of control, respectively, while norepinephrine (NE) concentration was depressed to 10 and 18% of control. These two respective dosages also decreased striatal tyrosine hydroxylase (TH) activity to 67 and 10% of control, respectively, while nigral TH activity was reduced to 59 and 20% of control. Striatal TPH activity was reduced to 74 and 81% of control, respectively, while the activity in the dorsal and median raphe remained unaltered. The intracerebroventricular administration of 1 mumol 2-hydroxy-4,5-methylenedioxymethamphetamine (6-OH-MDMA) failed to alter TPH activity, TH activity or NE concentration after 14 days. In contrast, 1 mumol of 2-hydroxy-4,5-methylenedioxyamphetamine (6-OH-MDA) induced a 30% increase in striatal TPH activity and a 50% increase in nigral TH activity. The study of the formation of 2,4,5-trihydroxyamphetamine after MDMA treatment may provide insight as to how MDMA destroys serotonergic nerve terminals.

Clinical evaluation of a naltrexone sustained-release preparation.

A clinical evaluation of the naltrexone bead, a biodegradable sustained-release dosage form of 3.0 mg in weight containing 70% naltrexone in a copolymer of lactic and glycolic acids, was carried out in 4 healthy normal males. Subjects were given an intravenous dose of 10 mg naltrexone and approx. 1 week later a 63-mg dose of naltrexone by subcutaneous administration of the beads. Challenge doses of 15 mg morphine were given to each subject during the study for the assessment of narcotic blockade effects of naltrexone. For a 2-4-week period after bead administration, relatively constant plasma levels were maintained at 0.30-0.46 ng/ml for naltrexone and were 0.64-1.07 ng/ml for naltrexol. Urine levels for unchanged and conjugated naltrexone were 79-215 ng/ml and for naltrexol were 315-500 ng/ml. From kinetic analysis, an average of 2.4-2.7% of implanted dose was absorbed each day from the administration of the beads. Opiate effects of morphine challenges were mitigated during the 2-4-week period after administration of naltrexone beads.

Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry.

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.

Clinical pharmacokinetics of escalating i.v. doses of dexanabinol (HU-211), a neuroprotectant agent, in normal volunteers.

The pharmacokinetics of dexanabinol (HU-211), a synthetic, nonpsychotropic cannabinoid with neuroprotectant action, was evaluated in a phase I clinical trial. The compound was administered at doses of 48 mg, 100 mg, and 200 mg as short i.v. infusions in a Cremophor-ethanol vehicle diluted with saline. All administrations were well-tolerated and no compound-related side-effects were observed. Plasma concentrations of dexanabinol were quantitated using a GC/MS/MS technique which provided a limit of quantitation of 100 pg/ml. The elimination of dexanabinol was best fitted to a 3-compartment model with a rapid distribution half-life (< 5 min), an intermediate phase half-life of approximately 90 min, and a slow terminal elimination half-life (approximately 9 h). The pharmacokinetics were linear over the evaluated dose range. The plasma clearance of the drug was high (1,700 ml/min) and the volume of distribution approximately 15 l/kg. These data are similar to those reported for naturally occurring cannabinoids such as delta 9-tetrahydrocannabinol and cannabidiol.

Ring formation in a pentapeptide with alternating L and D residues: an analogy to cyclization in the biosynthesis of peptide antibiotics.

Acetylation of L-isoleucyl-D-alanyl-D-alanyl-L-valyl-D-leucine with acetic anhydride followed by methylation with diazomethane yielded the expected acetylpentapeptide methyl ester with molecular weight 541, but also resulted in the formation of a by-product with molecular weight 555. The incorporation of the mass corresponding to CH2 seems to be due to ring closure--via a mixed anhydride--and methylation of the cyclol derivative thus formed. A preferred, ring-like conformation stabilized by intramolecular hydrogen bonds that in turn are the consequences of the alternation of D- and L- residues in the sequence, is invoked as explanation for the unexpected cyclization. This assumption is supported by the conversion of the pentapeptide methyl ester to desthiomalformain in molten imidazole.

Chemical-ionization mass spectrometry of beta-lactam antibiotics.

Chemical-ionization (CI) mass spectra are described for methyl esters of eight clinically significant penicillins and their breakdown products. The substances give spectra with very few fragment ions and contain easily discernible protonated molecule ions. The main cleavage reaction is postulated to involve a retro 2+2 Diels-Alder-type fragmentation of the beta-lactam ring liberating one fragment (m/e=174) that is characteristic of the penicillin nucleus and a second fragment that is molecule specific, as it contains the elements of the side chain. The other fragment ions, though interesting, are of minor intensity. The free acids, on the other hand, fragment more extensively because of their relative instability and lack of volatility. These spectra resemble electron impact spectra more closely and, though they encode more structural information, are less reproducible from run to run. The ease with which the esters can be made and the relative simplicity of their CI mass spectra make this method significant for the identification and characterization of beta-lactam antibiotics.

Comparative investigation of disposition of 3,4-(methylenedioxy)methamphetamine (MDMA) in the rat and the mouse by a capillary gas chromatography-mass spectrometry assay based on perfluorotributylamine-enhanced ammonia positive ion chemical ionization.

A gas chromatography-mass spectrometry assay based on perfluorotributylamine-enhanced ammonia positive ion chemical ionization has been developed for MDMA and three of its primary metabolites in biological specimens; the assay is linear from 2 to 1000 ng ml-1. Quantitatively, more of an administered dose of 10 mg kg-1 MDMA was excreted by the mouse (72%) than by the rat (35%); most in both species was excreted in urine and within 24 h. The difference in per cent excretion is entirely due to proportionally greater excretion of the parent drug by the mouse. 4-Hydroxy-3-methoxymethamphetamine (HMM) is the major urinary metabolite in both species. HMM and another primary metabolite, 4-hydroxy-3-methoxyamphetamine (HMA), were excreted mainly as glucuronide and sulphate conjugates (> 85%).

Measurement of lysergic acid diethylamide (LSD) in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry.

A previously reported procedure for quantification of LSD in urine was modified to permit measurement of the drug in plasma. After addition of deuterium-labelled LSD, the plasma is extracted and the extract is treated with trifluoroacetylimidazole to convert the LSD to its N-trifluoroacetyl derivative. The derivatized LSD is analyzed by capillary column gas chromatography/negative ion chemical ionization. Plasma fortified with known concentrations of LSD gave linear responses from 0.1 to 3.0 ng/mL with this assay. The method was used to determine pharmacokinetic parameters for LSD after oral administration (1 microgram/kg) to a male volunteer. The apparent plasma half-life was determined to be 5.1 h. The peak plasma concentration of 1.9 ng/mL occurred 3 h after administration.

Cocaine metabolism in man: identification of four previously unreported cocaine metabolites in human urine.

Cocaine and 11 of its metabolites were identified in a urine specimen from a cocaine user. Four of the metabolites are reported for the first time: ecgonidine, norecgonidine methyl ester, norecgonine methyl ester, and m-hydroxy-benzoylecgonine. The structures of the newly identified metabolites were confirmed by comparison of their gas chromatographic retention times and their electron ionization and chemical ionization mass spectra with the corresponding data obtained on synthesized standards. Other metabolites present were benzoylecgonine, ecgonine methyl ester, ecgonine, ecgonidine methyl ester, norcocaine, p-hydroxycocaine, and m-hydroxycocaine.

Comparison of a TLC method with EMIT and GC/MS for detection of cannabinoids in urine.

Urine specimens were analyzed in parallel with a new TLC method, an EMIT assay, and a reference GC/MS method. At a 9-carboxy-THC cutoff of 20 ng/mL, the TLC method correctly identified 92% of the positive urines and 97% of the negative urines. In contrast, only 63% of the urine specimens shown by GC/MS to contain greater than 20 ng/mL of 9-carboxy-THC were identified as positive by the EMIT d.a.u. assay at the 100-ng/mL cannabinoid cutoff.

Analysis of naltrexone and 6-beta-naltrexol in plasma and urine by gas chromatography/negative ion chemical ionization mass spectrometry.

A procedure for the analysis of naltrexone and 6-beta-naltrexol in plasma and urine samples is described. The method takes advantage of the specificity of negative ion chemical ionization mass spectrometry and the resolving power of capillary column chromatography to achieve a limit of quantitation of 0.1 ng/mL. The trideuterated analogs of naltrexone and 6-beta-naltrexol are used as internal standards. Samples are first made basic with K2HPO4 buffer (50% w/v), and then extracted twice with n-butyl chloride-acetonitrile (4:1). After back extraction into 0.2 N H2SO4, the samples are again extracted with n-butyl chloride-acetonitrile. The extracts are derivatized with 2% methoxyamine in pyridine and pentafluoropropionic anhydride to form the methoxime bis-(pentafluoropropionyl) derivative of naltrexone and the tris-(pentafluoropropionyl) derivative of 6-beta-naltrexol. The derivatized extracts are analyzed by selected ion monitoring of prominent ions formed by electron-capture negative ion chemical ionization.

Determination of naltrexone and 6-beta-naltrexol in plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Solid-phase extraction (SPE) and a one-step derivatization are combined with gas chromatography-negative ion chemical ionization-mass spectrometry to simplify a previously reported method for the determination of naltrexone and its metabolite, 6-beta-naltrexol, in human plasma. Deuterated isotopomers of naltrexone and 6-beta-naltrexol are used as internal standards. After SPE, the extracts are derivatized with pentafluoropropionic anhydride at room temperature to form predominantly the bispentafluoropropionyl derivative of naltrexone and the trispentafluoropropionyl derivative of 6-beta-naltrexol. The derivatized extracts are analyzed by monitoring ion currents at m/z 633 (naltrexone), m/z 636 (naltrexone-2H3), m/z 633 6-beta-naltrexol), and m/z 640 (6-beta-naltrexol-2H7). Control plasma samples containing 0.3, 3, or 30 ng/nl of each analyte were analyzed for precision and accuracy with the following results: intra-assay, the percentage of target concentrations were 107-113% for naltrexone and 107-120% for 6-beta-naltrexol, and the coefficients of variation (CVs) were 3.1-6.3% for naltrexone and 3.1-5.7% for 6-beta-naltrexol; interassay, the percentage of target concentrations were 103-110% for naltrexone and 110-113% for 6-beta-naltrexol, and the CVs were 6.1-9.1% for naltrexone and 5.9-9.1% for 6-beta-naltrexol. At the limit of quantitation (LOQ) of 0.1 ng/ml, both analytes quantified within 20% of the target concentration with CVs less than 17%. The extraction recoveries determined at 0.3 and 30 ng/ml were 79 and 80% for naltrexone and 76 and 75% for 6-beta-naltrexol. Bench-top stability tested with concentrations of 0.3 and 3.0 ng/ml did not decrease more than 10% from the zero-hour controls at 3, 6 and 24 h. Selectively was determined using plasma from six donors and none showed interfering peaks greater than 22% of the LOQ for naltrexone and 53% of the LOQ for 6-beta-naltrexol. Using this method, naltrexone and 6-beta-naltrexol were readily detected in plasma specimens collected 5.5 h after oral doses of 25 or 100 mg naltrexone. Following discontinuation of treatment, naltrexone was detected 30 h after the 100-mg dose, whereas 6-beta-naltrexol was detected 125 h after both the 25- and 100-mg doses.

Determination of methadone and its N-demethylation metabolites in biological specimens by GC-PICI-MS.

Methadone is often invoked for detoxification and maintenance of the opioid addict. We have developed and validated a sensitive and specific method for the analysis of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in human plasma, urine, and liver microsomes. This assay uses a solid-phase extraction. Separation and analysis of the analytes are performed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The protonated molecules (MH+) are monitored at m/z 264 and 267 for EMDP-d0 and -d3, m/z 278 and 281 for EDDP-d0 and -d3, and m/z 310 and 313 for methadone-d0 and -d3. The recovery of methadone and its metabolites exceeded 85% in the different matrices analyzed. Linear standard curves in plasma and in urine were obtained over the concentration range of 10-600 ng/mL (coefficients of determination: methadone, > or = 0.995; EMDP, > or = 0.994; and EDDP, > or = 0.996). With plasma and urine fortified at 25, 100, and 300 ng/mL, the assay was precise (intra-assay coefficients of variation [CVs], 2-12%; interassay CVs, 1-15%) and accurate (intra-assay percent of target, 85-107; interassay percent of target, 88-105) for all three analytes. Stability studies indicated that methadone and its metabolites are stable at room temperature in plasma and in urine for at least 1 week and in liver microsomes for at least 24 h. This method has now been shown to be useful for quantitation of methadone, EDDP, and EMDP in human urine and plasma and is also useful for quantitation of the amount of EDDP produced in human liver microsomes incubated with methadone. It provides an accurate and precise analytical tool for further studies on the metabolism of methadone.

Detection of LSD and metabolite in rat hair and human hair.

To examine the feasibility of detecting lysergic acid diethylamide (LSD) and its metabolites in hair, LSD was administered to rats with pigmented hair at 0.05, 0.1, 0.5, 1, and 2 mg/kg intraperitoneally once per day for 10 successive days. The rats were shaved just before the first administration, and newly grown hair was collected 4 weeks later. After being washed with 0.1% sodium dodecyl sulfonate and water and being dried in a desiccator, each 20-mg hair sample was extracted with 2 mliter methanol-5N HCl (20:1) under ultrasonication for 1 h and stored at room temperature for 14 h. The extract was evaporated to dryness, extracted from 0.1M NaOH with dichloromethane, and derivatized with a mixture of trimethylsilylimidazole, bis-(trimethylsilyl)acetamide, and trimethylchlorosilane (3:3:2, v/v/v) for gas chromatographic-mass spectrometric (GC-MS) analysis using LSD-d10 or lysergic acid methylpropylamide (LAMPA) as the internal standard. Selected ions were monitored at m/z 395, 293, and 279 for TMS-LSD and at m/z 381, 279, and 254 for the trimethylsilyl derivative of N-demethyl-LSD (TMS-norLSD). LSD and norLSD were also detected by high-performance liquid chromatography (HPLC) with fluorometric detection (excitation, 315 nm; emission, 420 nm). LSD was detected in the rat hair following the lowest dose (0.05 mg/kg), whereas norLSD was only detectable in the hair following the highest dose (2 mg/kg). The same GC-MS and HPLC assays were applied to the analysis of hair from 17 self-reported LSD users, and LSD was detected in two of the samples.

Determination of ibogaine and 12-hydroxy-ibogamine in plasma by gas chromatography-positive ion chemical ionization-mass spectrometry.

Ibogaine, an indolamine derivative, is currently being investigated as a potential agent in the treatment of stimulant and opiate addiction. We developed a rapid, sensitive, and specific method for the analysis of ibogaine and its putative active metabolite, 12-hydroxy-ibogamine (12-OH-ibogamine). This assay employs a one-step basic extraction with n-butyl chloride-acetonitrile (4:1), followed by derivatization of the metabolite using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. The derivatized extracts were analyzed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The ions monitored were at m/z 311, 314, and 411, which correspond to the protonated molecules (MH+) for ibogaine, ibogaine-d3, and 12-OH-ibogamine.tert-butyldimethylsilyl, respectively. Linear standard curves were obtained over the concentration range of 1 0-1 000 ng/mL (average r2, 0.995 for ibogaine and 0.992 for 12-OH-ibogamine; n = 3). Limits of quantitation were 10 ng/mL. The interrun and intrarun coefficients of variation for the assay of ibogaine at 25, 100, and 300 ng/mL ranged from 2.9 to 8.8%. We also established the extraction and chromatographic conditions to monitor the 12-hydroxylated metabolite. A suitable internal standard was not yet obtained so the method could only provide semiquantitative information for 12-OH-ibogamine. Chemical stability studies of these analytes indicated that ibogaine and 12-OH-ibogamine were stable in a human plasma matrix at room temperature for a period of at least 1 week.

Determination of LSD in urine by capillary column gas chromatography and electron impact mass spectrometry.

A procedure for the determination of LSD (lysergic acid diethylamide) in urine at concentrations as low as 0.5 ng/ml is presented. After addition of deuterium-labeled LSD as the internal standard, a rapid n-butyl chloride extraction of LSD from urine at pH 8 is followed by formation of the trimethylsilyl (TMS) derivative by treatment with N,O-bis(trimethylsilyl)trifluoroacetamide. The TMS derivative of LSD is identified and quantified by selected ion monitoring with a fused-silica capillary column and electron impact ionization. The procedure was used to monitor LSD concentrations in urine for eight hours following oral administration of 70.5 micrograms of LSD to two human volunteers. Concentrations of LSD determined by the assay are compared with concentrations determined by two other methods of analysis, a radioimmunoassay and a high-performance liquid chromatographic (HPLC) assay. Data concerning the stability of LSD in urine are also presented.