Simultaneous determination of delta9-tetrahydrocannabinol and 11-nor-9-carboxy-delta9-tetrahydrocannabinol in human plasma by solid-phase extraction and gas chromatography-negative ion chemical ionization-mass spectrometry.

Delta9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) in human plasma can be simultaneously detected using solid-phase extraction with gas chromatography and negative ion chemical ionization mass spectrometry. THC-d3 and THCA-d3 are added as internal standards; protein is precipitated with acetonitrile and the resulting supernatants diluted with 0.1 M sodium acetate (pH 7.0) prior to application to the solid-phase extraction columns. THC and THCA were eluted separately and then pooled, dried under air, and derivatized with trifluoroacetic anhydride and hexafluoroisopropanol. The derivatized THC-d0 gives abundant molecular anions (m/z 410), and the derivatized THCA-d0 gives abundant fragment ions (m/z 422) formed by loss of (CF3)2CHOH from its molecular anion. The recoveries of THC and THCA were 74% and 17%, respectively. The lower and upper limits of quantitation were 0.5 and 100 ng/mL for THC and 2.5 ng/mL and 100 ng/mL for THCA. The within-run accuracy and precision for THC (measured at 0.5, 1, 10 and 75 ng/mL) ranged from 98 to 106% (% target) and 4.1 to 9.5 (%CV), respectively. For THCA, the within-run accuracy and precision (measured at 2.5, 5, 10, and 75 ng/mL) ranged from 89 to 101% and 4.3 to 7.5%, respectively. The between-run accuracy and precision for THC ranged from 92 to 110% and 0.4 to 12.4%, respectively. The between-run accuracy and precision for THCA ranged from 97 to 103% and 6.5 to 12.3%, respectively. In processed samples stored in reconstituted form at -20 degrees C, THC and THCA were stable for at least three days. THC and THCA stored in plasma were stable following three freeze/thaw cycles. THC and THCA in whole blood at room temperature for 6 h, or in plasma stored at room temperature for 24 h, did not show significant change. Storage in polypropylene containers for 7 days at -20 degrees C and the presence of 1% sodium fluoride or the cannabinoid receptor antagonist, SR141716, at 1 microg/mL did not interfere with the quantitation of THC and THCA. In three individuals who smoked marijuana under controlled dosing conditions, peak THC concentrations of 151, 266, and 99 ng/mL were seen in the first plasma samples drawn immediately after the end of smoking, and corresponding peak THCA concentrations of 41, 52, and 17 ng/mL occurred at 0.33 to 1 h after cessation of smoking.

The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release.

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.

Determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in plasma after intravenous and intrathecal morphine administration using HPLC with electrospray ionization and tandem mass spectrometry.

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization (API) mass spectrometry (MS) has become a useful technique in the direct analysis of low concentrations of conjugated opiate metabolites. Previous methods using HPLC with traditional detection methods do not have the sensitivity to detect low concentrations of most conjugated drug metabolites. Methods using gas chromatography-mass spectrometry (GC-MS) require hydrolysis and derivatization of the sample followed by an indirect quantitation of conjugated metabolites. Recently, several reports have described direct analysis of opiates and their glucuronide conjugates by HPLC and API-MS. These methods report lower limits of detection than GC-MS methods and quantitation in the low nanogram-per-milliliter range for the glucuronide metabolites of morphine. This report describes an HPLC-electrospray-MS-MS method capable of detecting subnanogram concentrations of morphine (MOR) and its 3- and 6-glucuronide metabolites (M3G and M6G, respectively). The assay has a dynamic range of 250-10,000 pg/mL for M3G and M6G and 500-10,000 pg/mL for MOR. Inter- and intra-assay precision and accuracy varied by less than 8% for all analytes at 750-, 2500-, and 7500-pg/mL concentrations. This assay was used for the determination of MOR, M3G, and M6G in human plasma after intravenous (i.v.) and intrathecal (i.t.) administration of MOR and its effects on the ventilatory response to hypoxia. Peak plasma concentrations of MOR and M6G were measured 1 h after i.v. administration of MOR. Peak concentrations of M3G were measured 2 h after i.v. administration of MOR. After i.t. administration of MOR, peak concentrations of M3G were measured 8 h postdose. MOR was not detected in plasma of patients administered MOR i.t.. Subnanogram concentrations of M6G were measured in the plasma of five of nine patients administered MOR i.t..

Quantitation of alprazolam and alpha-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS.

A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and alpha-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 microL/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL for both the parent drug and metabolite and linear to 50 ng/mL. The intra-assay percent coefficients of variation (%CV) for AL at 2, 5, and 20 ng/mL were all < or = 5.6. At these concentrations, and all OH-AL intra-assay %CVs were < or = 8.4. The interassay variabilities for AL were 11.8%CV, 8.7%CV, and 8.7%CV at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL interassay variabilities were 9.6%CV, 9.2%CV, and 7.8%CV at the same concentrations, respectively. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/mL 1 h after the dose and AL was detectable for 48 h. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples.

Enantioselective gas chromatography-negative ion chemical ionization mass spectrometry for methylphenidate in human plasma.

Therapeutic doses of Ritalin, a racemic mixture of d- and l-threo-methyphenidate, result in low plasma concentrations of methylphenidate. In order to assess the safety and efficacy of methylphenidate, a sensitive analytical method is needed. A gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) assay capable of measuring both d- and l-enantiomers in human plasma was developed and validated to support clinical studies involving administration of d,l-methylphenidate. d,l-Methylphenidate-d3 is added to 1-mL plasma samples. The plasma samples are made basic, mixed with isopropanol and extracted with hexane. The hexane extracts are then back-extracted into 0.1 N HCl. The acidified aqueous extract is made basic, cooled to ice temperature, and the methylphenidate derivatized with heptafluorobutyryl-l-prolyl chloride. The two diastereomeric derivatives are then extracted into hexane. The hexane extract is evaporated to dryness, reconstituted in ethyl acetate, and analyzed by GC-NCI-MS. This method can accurately (+/- 5% target) and precisely (< 11.1% coefficient of variation) quantitate enantiomers of threo-methylphenidate in human plasma and in the whole blood at concentrations ranging from 0.75 to 100 ng/mL. Plasma samples are stable for up to five freeze-thaw cycles when the duration of each cycle did not exceed 0.5 h. The drug degraded gradually when plasma samples were left at room temperature; a 6% loss at 3 h progressed to 17% at 12 h and to 35% at 24 h. Therefore, it is important that extraction of plasma samples begins within 0.5 h after samples are removed from the freezer. Whole blood stability results show that concentrations of methylphenidate in whole blood, with or without NaF, stored for up to 6 h at room temperature did not deviate from the target concentration by more than 13%. The derivatized methylphenidate in extract is stable at 4 degrees C for up to 10 days.

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors.

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.

A comparison of ONTRAK TESTCUP, abuscreen ONTRAK, abuscreen ONLINE, and GC/MS urinalysis test results.

This study was designed to compare results obtained from two separate on-site drug testing kits (ONTRAK TESTCUP and Abuscreen ONTRAK) with those obtained from laboratory based immunoassay and GC/MS. Abuscreen ONLINE immunoassay was used to select 250 negative samples and 100 presumptive-positive samples each for cocaine/metabolites, opiates and cannabinoids. Presumptive-positive samples were selected if the immunoassay response was > or = 300 ng/mL for cocaine/metabolites (BZE), > or = 300 ng/mL for opiates or > or = 50 ng/mL for cannabinoids (THC-COOH). GC/MS was used to confirm that each selected sample contained > or = 150 ng/mL BZE, > or = 300 ng/mL morphine/codeine or > or = ng/mL THC-COOH. TESTCUP results had a 100% agreement with GC/MS and a > 99% agreement with ONLINE when testing negative samples. The agreement between TESTCUP and ONLINE results for samples containing opiates was 100%. Results of testing samples containing BZE with TESTCUP demonstrated a 98% agreement with both GC/MS and ONLINE. Both discrepant samples contained BZE at concentrations < or = 300 ng/mL. The least agreement between TESTCUP and ONLINE results was found when testing samples containing THC-COOH. The agreement with ONLINE and GC/MS was 92% and all discrepant samples had GC/MS determined THC-COOH concentrations less than 50 ng/mL. A 100% agreement was obtained between expected and recorded TESTCUP results for QC samples fortified to contained BZE, morphine or THC-COOH at concentrations within 120% of the screening cutoffs. ONTRAK had a 100% agreement with both GC/MS and ONLINE when testing negative samples and samples that contained opiates. ONTRAK had a 91% agreement with GC/MS and ONLINE for testing of samples that contained BZE. The least agreement between ONTRAK and ONLINE results was found when testing samples that contained THC-COOH. The agreement was 89%, however, all discrepant samples contained GC/MS concentrations of THC-COOH less that the 50 ng/mL cutoff. With ONTRAK, a 100% agreement was obtained between expected and recorded results QC samples that contained morphine or THC-COOH and a 97.7% agreement was obtained between expected and recorded results on QC samples that contained BZE.

Lack of long-term changes in cocaine and monoamine concentrations in rat CNS following chronic administration of cocaine.

In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d. for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.

Immunoassay detection of nordiazepam, triazolam, lorazepam, and alprazolam in blood.

The ability of commercial benzodiazepine immunoassays to detect nordiazepam, lorazepam, alprazolam, and triazolam in blood samples was investigated. Two radioimmunoassays (RIA) (Abuscreen [RIA-a] and the Diagnostic Products Corporation serum kit [RIA-d]), two enzyme immunoassays (EIA) (Emit d.a.u. [EIA-u] and Emit TOX serum assay [EIA-s]), and two fluorescence polarization immunoassays (FPIA) (X-systems urine [FPIA-u] and X-systems serum assay [FPIA-s]) were evaluated for their ability to detect benzodiazepines in fortified drug-free human or bovine blood. Prior extraction of the blood was necessary for analysis on the equipment used for EIA and FPIA. Extraction with an organic solvent, such as butyl chloride used in this study, was preferable to precipitation with methanol or zinc sulfate. For all these assays, extraction eliminated matrix effects and offered the possibility of increased sensitivity by reconstitution of the extract in a smaller volume. Extraction was necessary in bovine blood, as there is some nonextractable substance(s) in this matrix that increases non-specific binding. Using extraction with reconstitution in one-half the original volume, the apparent limit of detection for nordiazepam in blood ranged from 3 ng/mL, with RIA-d, to 30 ng/mL, with EIA-s and FPIA-s. These limits of detection were improved by further reduction of the reconstitution volume. RIA-a, EIA-s, and FPIA-s had cross-reactivity for alprazolam that was equivalent to or slightly better than nordiazepam. RIA-d had enhanced cross-reactivity for alprazolam at 30-300 ng/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.

Release of 3H-norepinephrine from adrenergic nerves in chick heart.

The metabolic disposition of 3H-norepinephrine (3H-NE) was studied in adrenergic nerves of the chick heart. Spontaneous overflow of 3H was associated with considerable metabolism of NE; about 90% of the 3H-NE appeared as acid and neutral metabolites. Overflow of 3H increased markedly during depolarization by K+ and over 70% of the 3H was unmetabolized 3H-NE. This 3H-NE seems to have been protected from intraneuronal metabolism (probably by vesicles) and to have been released by exocytosis. By contrast, tyramine-induced overflow included a large proportion of metabolites (approximately 70%) perhaps because of a nonexocytotic release mechanism similar to that in adult tissues.

Local anesthetics, mepacrine, and propranolol are antagonists of calmodulin.

Local anesthetics such as dibucaine, QX572, tetracaine, and phenacaine, as well as other drugs with local anesthetic-like properties (e.g., mepacrine, propranolol, and SKF 525A) inhibit the specific calmodulin-dependent stimulation of erythrocyte Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and cyclic nucleotide phosphodiesterases (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) from brain and heart. Basal activities of these enzymes in the absence of calmodulin are relatively unaffected by concentrations of local anesthetics that strongly inhibit the specific stimulation by calmodulin. Increasing calmodulin, but not Ca2+, overcomes the inhibitory action of the local anesthetics on brain phosphodiesterase. However, excess calmodulin does not fully restore activity of erythrocyte CA2+-stimulated ATPase. Although the mechanism(s) by which the local anesthetics act is unclear, they inhibit binding of 125I-labeled calmodulin to the erythrocyte membrane. Antagonism of calmodulin provides a molecular mechanism that may explain the inhibition of many Ca2+-dependent cellular processes by local anesthetics--e.g., Ca2+ transport, exocytosis, excitation-contraction coupling, non-muscle-cell motility, and aggregation.