Quantitative measurement of delta 9-tetrahydrocannabinol and two major metabolites in physiological specimens using capillary column gas chromatography negative ion chemical ionization mass spectrometry

delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.     

Brain microsomal oxidation of delta 8- and delta 9-tetrahydrocannabinol

Brain microsomes of mice, rats, guinea pigs and rabbits catalyzed the oxidation of delta 8- and delta 9-tetrahydrocannabinol to their monohydroxylated metabolites. The most prominent metabolite was the 4'-hydroxylated metabolite on the pentyl side chain of the cannabinoids in all species tested, except that the 5'-hydroxylation of delta 9-tetrahydrocannabinol was most abundant in the guinea pig. These results are quite different from the metabolic profile of the cannabinoids with hepatic microsomes.     

Stereospecific elimination of deuterium as a method for determining the stereochemistry of a number of metabolites of the tetrahydrocannabinols

The stereospecific elimination of the 3-deuterium atom from metabolites of [2H]-analogues of delta 1-tetrahydrocannabinol (delta 1-THC), delta 6-THC and delta 7-THC has been investigated as a possible method for determining the stereochemistry of metabolites substituted with hydroxy or acid groups in the terpene ring. Elimination of HCOOTMS was found to involve the 3-hydrogen of the axial but not the equatorial isomer of hexahydrocannabinol-7-oic acid, a metabolite of all three cannabinoids. Similar stereospecific eliminations were observed during the loss of TMSOH from the TMS derivatives of 5 alpha-hydroxy-delta 6-THC, 6 alpha-hydroxy-delta 1-THC and 1 alpha, 2 beta-dihydroxy-delta 1-THC. Loss of TMSOH from 1 alpha, 7- and 1 beta, 7-dihydroxy-HHC involved the 3-hydrogen in both cases but the isomers could be distinguished as their alkane-boronate derivatives; only the derivative of 1 alpha, 7-dihydroxy-HHC lost the boronate ring with stereospecific removal of the 3-hydrogen. The stereochemistry of the four isomers of 1,6-dihydroxy-HHC could not be determined in this way as the [M-TMSOH]+ ions from all four compounds had lost the 3-hydrogen, presumably as the result of 1,6-bond cleavage.     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

Mass spectrometry of dimethylthiophosphinates of aromatic hydroxy compounds

The mass spectra of 20 differently substituted dimethylthiophosphinic esters of aromatic hydroxy compounds are presented. Fragmentation routes were investigated using high resolution mass measurements, decoupled metastable determinations and deuterium labelling. All compounds exhibited abundant molecular ions and typical phosphorus-containing ions. Characteristic elimination processes strongly dependent upon the respective type of substitution were observed. Due to their high stability, their great ease of formation and their good gas chromatographic properties these new types of derivatives are of special interest for establishing gas chromatography mass spectrometry profiles of acidic catecholamine metabolites.     

Cardiovascular effects of delta 9- and delta 9(11)-tetrahydrocannabinol and their interaction with epinephrine

The administration of delta-9-tetrahydrocannabinol (delta 9-THC, 0.078-5.0 mg/kg, i.v.) to rats anesthetized with pentobarbital caused as much as a 50% decrease in mean arterial blood pressure, heart rate and respiratory rate in a dose-dependent manner. Delta-9(11)-tetrahydrocannabinol (delta 9(11)-THC) was approximately 8-fold less potent than delta 9-THC in its hypotensive effect and had smaller effects on heart and respiratory rates that were not dose-related at doses below 5 mg/kg. Alternate injections of epinephrine (2 micrograms/kg) with vehicle and increasing cannabinoid doses (1.25-5.0 mg/kg) indicated a potentiation of both the duration of the pressor effect and the magnitude of the reflex bradycardic effect of epinephrine by both delta 9- and delta 9(11)-THC. Epinephrine also produced arrhythmias in rats receiving cannabinoids, but not in rats receiving alternate injections of vehicle. It is concluded that both cannabinoids have adverse effects on the cardiovascular system and adverse interactions with epinephrine in rats anesthetized with pentobarbital.     

Comparison of gas chromatography mass spectrometry methods for the determination of delta9-tetrahydrocannabinol in plasma

A method for the identification of delta9-tetrahydrocannabinol by gas chromatography mass spectrometry has been developed, and this method has been compared with other techniques, such as detection via thin-layer chromatography using tritium labeled delta9-tetrahydrocannibinol and a dual gas chromatographic method. The gas chromatographic mass spectrometric method was found to be equal or superior to other techniques and has the added advantage of being highly specific for the compound analyzed. An alternate approach using chemical ionization is also described; however, this procedure does not show significant advantages over the electron impact method. These methods show a practical lower detection limit of 500 pg ml-1 of plasma in clinical practice.     

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.     

Failure of acute and chronic administration of delta 9-tetrahydrocannabinol to affect the repeated acquisition of serial position response in pigeons

Pigeons were trained to acquire a new four-response position sequence each day by pecking three response keys in a predetermined order. The key color varied after each correct response prior to food delivery. Acute administration of delta 9-tetrahydrocannabinol (delta 9-THC) up to a dose that completely eliminated responding, had no effect on total acquisition errors, or on within session patterns of error elimination. Chronic administration of delta 9-THC (3-10 mg/kg/day), either before or after the session for 4-7 weeks, also did not affect these error measures, although rates of responding were markedly suppressed and at times no responding occurred. Discontinuation of delta 9-THC administration for periods of 4-6 weeks also was without effect on errors. These experiments suggest that neither acute nor chronic delta 9-THC produce specific effects on the repeated acquisition of serial position responses in pigeons.     

Elevation of brain prostaglandin E2 levels in rodents by delta 1-tetrahydrocannabinol.

An isotopic dilution procedure using specific prostaglandin E2 (PGE2) brain receptors was utilized to determine the changes in brain PGE2 levels subsequent to drug exposure. Delta-1-tetrahydrocannabinol (delta 1-THC) stimulated PGE2 synthesis resulting in increased brain concentrations when compared with vehicle treated rats and mice. Indomethacin markedly inhibited the delta 1-THC elevated rise in PGE2 levels presumably by inhibition of prostaglandin synthetase. The delta 1-THC-induced increase in PGE2 brain levels was also suppressed by i.v. administered rabbit PGE2-antiserum. This suggests that one of the sites of delta 1-THC action is extracerebral and from here a portion of the released prostaglandins are transported to the brain. These results add further support to previous data that delta 1-THC given orally results in an increase in brain PGE2 levels.     

The nature of the inhibition of cholesterol esterase by delta 1-tetrahydrocannabinol

An improved assay for cholesterol esterase based on the use of fatty acid radiolabelled cholesterol esters has been developed. The method was used to demonstrate the effects of delta 1-tetrahydrocannabinol on a crude Leydig cell esterase preparation and on crystalline pancreatic esterase. Both enzymes were inhibited and the Km values determined (6.6 mumol/1 for the Leydig cell esterase and 6.25 mumol/1 for the pancreatic enzyme). While the former exhibited a mixed type of inhibition, the latter clearly was competitive.     

Liquid chromatographic-mass spectrometric quantitation of Delta9-tetrahydrocannabinol and two metabolites in pharmacokinetic study plasma samples

A sensitive method for the determination of Delta(9)-tetrahydrocannabinol and its metabolites, 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid and 11-hydroxy-Delta(9)-tetrahydrocannabinol, in rat and guinea pig plasma was developed using high-performance liquid chromatographic separation with electrospray ionization mass spectrometry detection and a simple liquid-liquid extraction technique. The mean recoveries for Delta(9)-tetrahydrocannabinol, 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid, and 11-hydroxy-Delta(9)-tetrahydrocannabinol were 96, 92, and 85%, respectively. The lower limit of quantification (LLOQ) for all three compounds was 5 ng/ml and the limit of detection (LOD) was 2 ng/ml. This assay method utilizes the increased sensitivity and selectivity of mass spectrometric (MS) detection and a simple extraction step for the determination of Delta(9)-tetrahydrocannabinol and its metabolites in plasma, and thus yields a more efficient pharmacokinetic analysis method than has previously been described.     

Radical peroxidation of palmitoyl-lineloyl-glycerophosphocholine liposomes: Identification of long-chain oxidised products by liquid chromatography-tandem mass spectrometry

Liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was used to identify palmitoyl-lineloyl-glycerophosphatidylcholine oxidation products (PL(O(1-6))PC). Structural and positional isomers of keto, hydroxy and/or epoxy, and hydroperoxide derivatives of PLPC were identified based on MS/MS data, namely product ions attributed to lyso-phosphatidylcholines, product ions formed by loss of nH(2)O and H(2)O(2) from [MH](+) ions groups, and product ions involving the hydroxy groups, providing information about the position of these groups and of the double bonds along the carbon chain of lineloyl moiety.     

Identification of hydroxylated chlorodibenzo-p-dioxins, chlorodibenzofurans, chlorodiphenyl ethers and chloronaphthalenes as their methyl ethers by gas chromatography mass spectrometry

The methyl ethers of a number of hydroxylated (poly)chlorodibenzo-p-dioxins, chlorodibenzofurans, chlorodiphenyl ethers and chloronaphthalenes, representing all different hydroxy substitutions, were synthesized and their mass spectra investigated. With the exception of the methoxy derivatives of the chlorodibenzofurans, it appeared that the mass fragmentation patterns of the structural isomers of each class of compounds were very specific and allowed unambiguous assignment of the position of the methoxy group in the molecule. The different fragmentation patterns can be explained in terms of plausible mechanisms resulting in stable charge delocalized (oxonium) ions. Because of its diagnostic value, this method is useful in the structure elucidation of hydroxylated metabolites of pure isomers of chlorodibenzo-p-dioxins, chlorodiphenyl ethers and chloronaphthalenes.     

Novel conformationally restricted tetracyclic analogs of delta8-tetrahydrocannabinol.

Novel analogs of (-)-delta8-tetrahydrocannabinol (delta8-THC) in which the conformation of the side chain was restricted by incorporating the first one or two carbons into a six membered ring fused with the aromatic phenolic A ring were synthesized. The affinities of the novel ligands for CB1 and CB2 indicated that the "southbound" chain conformer retained the highest affinity for both receptors.     

delta 9-Tetrahydrocannabinol elicited ipsilateral circling behavior in rats with unilateral nigral lesion

The present study was designed to examine the influence of delta 9-tetrahydrocannabinol (THC) on the central dopaminergic system using circling behavior. THC 5 mg/kg i.p. produced ipsilateral circling in rats with unilateral nigral lesion by 6-hydroxy-dopamine. THC-induced ipsilateral circling was completely antagonized by 0.2 mg/kg of haloperidol. These findings suggest that THC may cause a presynaptic stimulation of nigrostriatal dopaminergic neurons.     

Use of Fourier transform 1H NMR in the identification of vitamin D2 metabolites

The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.     

delta 12-Prostaglandin J2, an ultimate metabolite of prostaglandin D2 exerting cell growth inhibition

L-1210 murine leukemia cells were exposed to prostaglandin D2 (PGD2), 10 micrograms/ml, in culture medium for various time, and subsequent cell growth was observed. More than 24 h exposure to PGD2 was required to inhibit cell growth almost completely. During this period, PGD2 degraded time-dependently into several products. The major product was identified as delta 12-PGJ2 by TLC, UV and mass spectra. When delta 12-PGJ2 was added to cells instead of PGD2, it evoked growth inhibition with much shorter contact time than PGD2. In addition, when the medium containing PGD2 was preincubated at 37 degrees C for 24 h, it elicited growth inhibition with only 6 h contact with cells. Furthermore, when the medium containing PGD2 was changed every 6 h during 24 h exposure time to cells, no significant growth inhibition was observed. These results suggested that PGD2 per se has little, if any, growth inhibitory activity, and delta 12-PGJ2 is an ultimate metabolite exerting growth inhibition. This action appears to be independent of cAMP, since delta 12-PGJ2 was virtually inactive in raising intracellular cAMP levels.     

NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa

The complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid, cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical shift of H-1" of the C5 side chain and 2'-OH. It was also observed that carboxylic acid substitution reduces intermolecular hydrogen bonding resulting in a sharpening of the H-5' signal in cannabinolic acid in deuterated chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3' and C-4' of cannflavin A and B were determined by HMBC spectra.     

Effect of delta 9-tetrahydrocannabinol on herpes simplex virus type 2 vaginal infection in the guinea pig

The present investigation was undertaken to determine whether delta 9-tetrahydrocannabinol (delta 9-THC) decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. The guinea pig was selected as the host since it has been shown to express a spectrum of primary herpes genitalis which is similar to that in humans. Animals were administered delta 9-THC or vehicle intraperitoneally on Days 1-4, 8-11, and 15-18. Herpes simplex virus was introduced intravaginally on Day 2. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding, and animal mortalities. Virus-infected animals treated with drug at doses of 4 and 10 mg/kg exhibited significantly greater severity of genital disease during the 30-day period of study when compared to virus-inoculated vehicle controls. A direct relationship was noted between dose of delta 9-THC and cumulative mortalities on Day 14 following primary infection. These results indicate that delta 9-THC decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig.