Cannabinoids with a propyl side chain in cannabis: occurrence and chromatographic behavior.

Neutral cannabinoids with a pentyl side chain-for example, cannabidiol, tetrahydrocannabinol, and cannabinol-are generally accompanied by homologs with a propyl side chain, of which at least one has psychotropic activity. Samples of hashish and marihuana from Asia especially sometimes have abundant amounts of propyl cannabinoids, the quantities being of the same order as that of the accompanying pentyl cannabinoids. Detection and identification of the propyl and pentyl cannabinoids in gas chromatography and thin-layer chromatography is discussed.

Kinetics of carbamazepine and carbamazepine-epoxide, determined by use of plasma and saliva.

The concentration-time curves of carbamazepine (CBZ) and its metabolite (carbamazepine-10,11-epoxide; CBZ-epoxide) were determined in patients undergoing long-term antiepileptic drug treatment with the use of plasma and saliva data. Plasma and saliva samples were assayed concurrently for each patient by liquid chromatography. There was excellent linear correlation between CBZ levels in saliva and plasma (r = 0.991, p less than 0.001) over a large concentration range. The saliva/plasma ratio for CBZ concentration was 0.26 +/- 0.01 (SD). Since CBZ binding to plasma proteins is in the order of 76%, saliva CBZ concentration seems to reflect the unbound fraction of the drug in plasma. CBZ-epoxide has not been detected in saliva. The pharmacokinetic parameters of CBZ-epoxide were determined in 6 patients. The pharmacokinetic parameters of CBZ obtained from saliva concentrations were in excellent agreement with those obtained from plasma concentrations. Thus, CBZ determination in saliva is convenient for controlling blood levels in patients as well as for studying pharmacokinetics. The half-life, the relative body clearance of CBZ, and the metabolite concentration during steady-state, expressed as percent the parent compound, appear to be significantly different in patients on single and combined drug therapy.

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function.

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.

The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes.

We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepine exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 microM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors.

Relationship between carbamazepine concentrations in plasma and saliva in man as determined by liquid chromatography.

The concentration of carbamazepine in plasma and saliva was determined in 7 subjects receiving carbamazepine. Plasma and saliva samples were assayed was an excellent linear relationship between carbamazepine concentrations in saliva and plasma (r = 0.991, p less than 0.001) over a plasma concentration range of 0.2--8.0 microgram/ml. The saliva/plasma ratio for carbamazepine was 0.26 +/- 0.02 (S.D.). Since carbamazepine binding to plasma proteins is in the order of 75%, the saliva concentration seems to reflect the concentration of the free drug in plasma. There is very little intrasubject and intersubject variation in the concentration ratio of carbamazepine. This study demonstrates that carbamazepine determination in saliva is a convenient method for optimizing blood levels of patients as well as for the study of pharmacokinetic properties.

Dynamically coated capillaries improve the identification power of capillary zone electrophoresis for basic drugs in toxicological analysis.

In systematic toxicological analysis (STA), analytical methods should have a high identification power. This can be suitably expressed by parameters such as mean list length (MLL) or discriminating power (DP). The reproducibility of a method has a great impact on its identification power, and should be as high as possible. In this study, two separation methods based on capillary zone electrophoresis (CZE) were evaluated towards STA applications. Besides a normal phosphate buffer, the commercially available buffer CElixir was used, which is a double-layer dynamic coating system. The coating stabilizes the endoosmotic flow, is independent of the pH, and is claimed to be more reproducible and faster at low pH than with normal buffers. A test set of 73 basic pharmaceutical compounds was analyzed by the two CZE methods. The total analysis time, including rinsing steps, was 8 min when the coating was used and 18 min without the coating. Effective mobilities were calculated and the reproducibilities were a factor of 2 better when the coating was used (between-days SD 0.020 and 0.040 m2/V s with and without the coating, respectively). MLL and DP were calculated for the two CZE methods and for combinations with standardized liquid and gas chromatography systems. CZE with CElixir coating clearly has a high potential for STA applications, as it was shown to have a higher identification power and shorter analysis times than normal CZE.

Size-exclusion chromatographic reconstitution of the bovine brain benzodiazepine receptor. Effects of lipid environment on the binding characteristics.

The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (Kd) of 2.7 +/- 1.2 nM and with 0.40 +/- 0.04 pmol binding sites per mg protein (Bmax). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (Kd 1.4-4 nM). Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at -20 degrees C or -80 degrees C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and Bmax over a period of 2 months.

Coupling device for desorption of drugs from solid-phase extraction-pipette tips and on-line gas chromatographic analysis.

Solid-phase extraction-pipette tips were used for micro solid-phase extraction of lidocaine and diazepam. Off-line desorption was done after in-vial collection for reference purposes, whereas with on-line desorption the eluate was directly introduced in the gas chromatograph. With both methods the total eluate (100 microl) was introduced into the GC system, which was equipped with a programmed-temperature vaporiser (PTV) for large volume injection. For on-line desorption a laboratory-made coupling device was developed to connect the pipette tips with the injector of the PTV. The coupling device was applied successfully since no leakage occurred at the connection of the coupling device and the pipette tip. No significant differences in recovery of lidocaine and diazepam and in presence of impurities were observed between chromatograms obtained with either off-line or on-line desorption. Preliminary experiments with standard solutions showed recoveries of about 75% for a concentration level of 1 microg/ml. The system seems particularly suitable for high-throughput analysis.

Toxicological analysis of whole blood samples by means of Bond-Elut Certify columns and gas chromatography with nitrogen-phosphorus detection.

The application of Bond-Elut Certify solid-phase extraction columns to the systematic toxicological analysis of whole blood was evaluated. The reproducibility of the extraction was tested with thirteen drugs varying in physico-chemical properties. Analysis was performed with capillary gas chromatography with nitrogen-selective detection. The recoveries were reproducible, as long as other limiting factors, e.g., chromatographic behaviour or volatility, do not play a significant role. The effect of limiting chromatographic behaviour was studied in more detail with the more sensitive mass spectrometry with selected-ion monitoring after converting the extracted morphine into its ditrimethylsilyl derivative.

Direct enantiomeric separation of mianserin and 6-azamianserin derivatives using chiral stationary phases.

The direct enantiomeric separation of mianserin and 6-azamianserin and some of their derivatives, respectively, by means of HPLC using two different chiral selectors was investigated. For the cellulose-based Chiralcel OD column, a strong dependence of the lipophilicity of the compounds tested on the retention behaviour was observed. To some extent, this was also found for the enantiomeric separation on the amylose-based Chiralpak AD column. In some cases a complementary behaviour of these two phases was observed: racemic mixtures that could not be separated by one column could be resolved by the other one.

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis.

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.

Feasibility of the direct coupling of solid-phase extraction-pipette tips with a programmed-temperature vaporiser for gas chromatographic analysis of drugs in plasma.

Solid-phase extraction-pipette tips (SPE-PTs) were used for micro solid-phase extraction of lidocaine and diazepam from plasma. Off-line extraction was followed by on-line desorption. On-line desorption was carried out by direct coupling of the SPE-PTs with the liner of the programmed-temperature vaporiser. This coupling only required shortening of the liner by maximally 16 mm, cutting the SPE-PT, and equipping the remaining part with two O-rings. Due to the heating of the injector the SPE-PTs were heated as well, which resulted in a significant amount of impurities. Pre-heating and pre-washing was performed prior to the extraction to reduce the impurity level. The internal coupling device was applied successfully for the analysis of plasma samples with gas chromatography (GC) and mass-selective detection. Detection limits of 0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam, respectively, using 200 microl plasma. Recoveries for both compounds were about 80%. Although it is possible, the internal coupling device was not developed to be used as such. The main goal of this coupling was to show the feasibility of the integration of SPE-PTs with GC and to realize an important step to new automated SPE-GC systems.

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. III. Determination of prednisolone in serum.

Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.

Recent developments in analytical toxicology: for better or for worse.

When considering the state of the art in toxicology from an analytical perspective, the key developments relate to three major areas. (1) Forensic horizon: Today forensic analysis has broadened its scope dramatically, to include workplace toxicology, drug abuse testing, drugs and driving, doping, environmental and veterinary toxicology, etc. (2) Basic analytical issues, focusing on a strongly widening array of relevant substances and metabolites at ever decreasing levels in a large variety of matrices. (3) Validation and interpretation: Because forensic analyses may have severe, punitive consequences, validation of methods and approaches plus proficiency testing are of utmost importance. Also, interpretation of the analytical results must be done with prudence in view of chemical and biological diversity in society. This review discusses the various pros and cons in these developments.

Pharmacokinetics of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin in freely moving rats.

The pharmacokinetics of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (1, N-0923) after intravenous, intraperitoneal, and oral administration was studied in freely moving male and female albino Wistar rats. In all cases, the dose was 10 mumol.kg-1. Levels of 1 in plasma were monitored up to 6 h after dosing by high-performance liquid chromatography. A biphasic disappearance pattern with a rapid distribution phase was observed in the curve of concentration in plasma versus time. Pharmacokinetic analysis based on a two-compartment model with elimination from the central compartment yielded the following parameters for male rats (mean +/- standard deviation, n = 3): elimination half-life was 108 +/- 7 min, apparent volume of distribution of the central compartment was 397 +/- 44 mL.kg-1, the plasma clearance was 32 +/- 4 mL.min-1.kg-1, and the apparent volume of distribution at steady state was 2.29 +/- 0.25 L.kg-1. No significant difference (analysis of variance p value > 0.25) was found in the pharmacokinetic data between male and female rats. An extremely fast absorption was found after intraperitoneal administration. Maximal concentrations in plasma were often observed at the first time point 5 min after dosing. The bioavailability (mean +/- standard deviation, n = 3) was 7.9 +/- 2.7% for male rats and 6.5 +/- 2.1% for female rats. An increase of the elimination half-life of at least 40% for male rats and 300% for female rats was observed, indicating dose-dependent kinetics after intraperitoneal administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Percutaneous absorption, metabolic profiling, and excretion of the penetration enhancer azone after multiple dosing of an azone-containing triamcinolone acetonide cream in humans.

Radioactive Azone (1.6%; 1-dodecylazacycloheptan-2-one) was incorporated in a therapeutic formulation containing triamcinolone acetonide at a concentration of 0.05%. This cream (TAZ) was applied for four consecutive days to human volunteers on the same 24-cm2 application area on the forearm for 12 h under occlusion. The percutaneous absorption of Azone as measured in the excreta appeared to be only 3.47 +/- 0.33% during the whole study period. Azone-derived radioactivity was predominantly excreted by the kidneys (97.8 +/- 0.4%). From the urinary excretion plot, it could be deduced that the flux of Azone through human skin increased during the study period, reaching a plateau within 2-3 d. Accumulation of Azone in the stratum corneum did not occur. Only unchanged Azone could be detected in the stratum corneum. Excretion was mainly in the form of very polar metabolites. Compared with pure Azone, the therapeutic formulation did not influence the metabolism, excretion route, or urinary elimination rate of the penetration enhancer.

Pharmacokinetics of clomipramine in depressive patients.

Ten patients with a vital depressive syndrome were treated for 4 weeks with clomipramine (CI), five receiving the drug by mouth and five receiving it first intramuscularly and then by mouth. Plasma concentrations of CI and desmethylclomipramine (DCI) were measured daily. Both concentrations showed marked interindividual differences, especially after oral administration of the drug. The mean relative CI clearance after repeated i.m. injections was 0.441 kg-1 hour-1. The route of administration proved to exert a marked influence on the plasma concentration ratio CI/DCI. In this small population, no significant relationship could be demonstrated between plasma CI and DCI concentrations, or the sum of both, and the clinical effect.

First-pass effect after rectal administration of thiazinamium methylsulfate.

The absorption and metabolism of the quaternary ammonium compound thiazinamium methylsulfate were studied in humans using plasma concentration data and urinary excretion measurements. After giving a dose of 150 mg in suppositories, the relative bioavailability was 5.8 +/- 3.2 (SD) % of the dose, comparable to the values obtained following oral administration. The degree of first-pass effect observed after rectal administration was comparable with that after oral administration.

Centrifugation or filtration in quantitative radioreceptor assays.

In quantitative radioreceptor assays the amount of a drug present in the medium to be assayed is inversely related to the amount of receptor-bound radiolabelled ligand. Usually, separation of the bound and free fractions of radiolabelled ligand is done by filtration, in which the bound fraction can easily be collected. However, the filtration disturbs the equilibrium between bound and free fractions, which may lead to erroneous results. Because the decrease in bound radiolabelled ligand is accompanied by an increase in free labelled ligand, we decided also to measure this free fraction after separation by centrifugation and to compare these data with the filtration data. In these experiments a radioreceptor assay for anticholinergics was employed. The results indicate that both methods are compatible in precision when appropriate conditions are used whereas each method has its specific features.

Chromatography in analytical toxicology--state of the art and future perspectives.

The aims of systematic toxicological analysis are considered with respect to the principal techniques available for quantitative and qualitative purposes. The use of thin-layer (TLC), gas-liquid (GLC), capillary GLC and high-performance liquid chromatography (HPLC) is discussed, with special reference to the need for standardized systems for toxicological analysis. The selectivity of correlations presented as mean list length values, derived from archives of Kováts retention indices in GLC and of Rf-values in up to eight TLC systems, is illustrated by some typical examples. The dependence of Kováts retention indices on column packing materials and on load capacity in CGLC is discussed and the particular difficulties of comparable standard systems in HPLC are considered. Conclusions are drawn concerning the established 'workhorse' techniques, such as TLC and GLC. The need to adapt and standardize other powerful analytical methods, such as HPLC, MS and GLC-MS, is examined with regard to the requirements of toxicological analysis.