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.

Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis.

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation. The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds. For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction. The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.

Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers.

Polymers imprinted with clenbuterol were used to study the influence of various post-polymerization treatments [e.g., thermal annealing, microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid template desorption] on the bleeding of residual template. The aim of the study was to reduce the bleeding to levels that would allow the use of the materials as affinity phases for extraction of clenbuterol from bovine urine at concentrations below 1 ng ml-1. After treatment, the clenbuterol imprinted polymers were packed into solid-phase extraction columns and the bleeding was estimated by quantifying the amount of template released in 10 ml of methanol-acetic acid (9 + 1 v/v). This was followed by an assessment of selectivity and recovery in comparison with non-treated material. The lowest bleeding level was found after MAE using 100% trifluoroacetic acid for 3 x 20 min at 100 degrees C. The collected eluate contained in this case 3 ng ml-1 of clenbuterol. The same material was subsequently used for the extraction of clenbuterol from spiked bovine urine. The resulting selectivity and recovery were lower compared with those obtained using the untreated material. A milder but still efficient method to reduce the bleeding level was found to be MAE with formic acid. In this case a bleeding level of 14 ng ml-1 was found after only a 1 h extraction time. In a second model system, using a polymer imprinted with L-phenylalanine anilide, the bleeding was reduced to a similar level by extensive on-line washing in good swelling solvents containing acid or base additives and after thermal annealing of the polymers in the dry state.

Fibers coated with molecularly imprinted polymers for solid-phase microextraction.

The simplicity and flexibility of solid-phase microextraction have been combined with the selectivity of molecularly imprinted polymers (MIPs). Silica fibers were coated reproducible with a 75-microm layer of methacrylate polymer either nonimprinted or imprinted with clenbuterol to compare their extraction characteristics under various conditions. Although the template molecule could be removed effectively from the imprinted polymer, structural analogues of clenbuterol were used for evaluation. The influence of pH on the extractability of brombuterol was investigated. Extraction yields up to approximately 80% were obtained when both types of fibers were used to extract brombuterol from phosphate buffer (pH 7.0). In contrast, yields of about 75 and <5% were obtained when extraction was performed from acetonitrile with imprinted and nonimprinted polymers, respectively, which demonstrates the selectivity of the MIP-coated fiber. Time sorption profiles were measured for the extraction of brombuterol from buffer and acetonitrile at the 10 and 100 ng/mL level with both types of fibers in order to compare extraction characteristics. Equilibrium times of about 30 and 90 min were found for the extraction of brombuterol from acetonitrile and buffer, respectively. The MIP-coated fibers were capable of extracting five structural analogues of clenbuterol from both buffer and acetonitrile, which suggests that the amine alcohol part of these molecules is responsible for interaction with the imprinted polymer. To achieve selective extraction of brombuterol from human urine, MIP-coated fibers were washed with acetonitrile after the extraction. Clean extracts and yields of approximately 45% were obtained, demonstrating the suitability of MIP-coated fibers for the analysis of biological samples.

A fluorescent receptor assay for benzodiazepines using coumarin-labeled desethylflumazenil as ligand.

This article describes a novel nonisotopic receptor assay for benzodiazepines with fluorescence detection. As labeled ligand (coumarin-labeled desethylflumazenil, CLDEF), a metabolite of the benzodiazepine antagonist flumazenil (desetheylflumazenil, Ro15-3890) has been coupled to a coumarin fluorophore, via a spacer. CLDEF had a Ki of 6.5 nM. To avoid the interference of the background fluorescence of the receptors in the measurement step, the bound CLDEF was dissociated from the receptors after the filtration step. This dissociation was achieved by incubating the CLDEF-bound to the receptors on the filters-with a weakly acetate buffer. The second filtrates then contained the previously bound CLDEF, which was then quantitated with a RP-HPLC system with a fluorescence detector. The results with a fluorescent receptor assay were very similar to those with a radioreceptor assay, in that the IC50 values of lorazepam were 7.2 +/- 0.5 and 6.6 +/- 0.7 nM, respectively.

Determination of clenbuterol in bovine liver by combining matrix solid-phase dispersion and molecular imprinted solid-phase extraction followed by liquid chromatography/electrospray ion trap multiple-stage mass spectrometry.

Matrix solid-phase dispersion (MSPD) is a new sample pretreatment for solid samples. This technique greatly simplifies sample pretreatment but, nonetheless, the extracts often still require an extra cleanup step that is both laborious and time-consuming. The potential of combining MSPD with molecularly imprinted solid-phase extraction (MISPE) was investigated in this study. Liver samples were ground in a mortar with C18 sorbent and the homogenized mixture packed into an SPE cartridge and placed on top of a MISPE cartridge. Subsequently, clenbuterol was eluted from the MSPD cartridge onto the MISPE cartridge using acetonitrile containing 1% acetic acid. The ability of the molecularly imprinted polymer to selectively adsorb analyte in acetonitrile was exploited for re-extracting clenbuterol directly from this acetonitrile extract via the double cartridge tandem system. The analyte was eluted from the MISPE cartridge using acidified methanol. A clear eluate was obtained, which was subsequently evaporated, redissolved, and analyzed by HPLC electrochemical detection (ECD) or ion trap mass spectrometry (LC/IT-MS). The MISPE cartridge used in this study was imprinted using bromoclenbuterol, a structural analogue of clenbuterol, as the template. These MISPE cartridges showed excellent stability. The complete extraction procedure was rapid, and recoveries exceeded 90% for the target analyte. The method detection limit for the LC/IT-MS procedure was < 0.1 microg/kg. This method, therefore, satisfies the stringent requirements of European Union regulation EEC 2377/90.

Enantioseparation of ofloxacin in urine by capillary electrokinetic chromatography using charged cyclodextrins as chiral selectors and assessment of enantioconversion.

A method was developed for the enantioseparation of ofloxacin, a member of the fluoroquinolones, using an anionic cyclodextrin-derivative with or without combination with a neutral cyclodextrin-derivative, as the chiral selector (s) in an electrokinetic chromatography system. The best results were obtained with 0.35 mM sulfated beta-cyclodextrin dissolved in a 50 mM phosphate buffer, pH 2.5, and at 15 degrees C. Under these conditions, a resolution of 2 was readily achieved. Furthermore, under adequate separation conditions, studies were performed in order to assess possible in vitro and in vivo enantioconversion of levofloxacin. The current method allows detection of 2 microg R-(+)-ofloxacine/mL diluted urine without the necessity of sample cleanup.

Use of molecularly imprinted solid-phase extraction for the selective clean-up of clenbuterol from calf urine.

A feasibility study was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for clean-up of clenbuterol from urine. A binding study of clenbuterol in several solvents was performed on a clenbuterol imprinted polymer as well as on a blank polymer. These binding experiments were used to find suitable loading, washing and elution solvents for the MISPE procedure. Extraction of clenbuterol from calf urine was performed by directly loading a 10-ml urine sample onto the MIP column. Thereafter the column was washed with 10 ml of acetonitrile containing 1% acetic acid, and finally clenbuterol was eluted with 6 ml of methanol containing 10% acetic acid. A recovery of 65% was obtained. This recovery could be increased up to 75% if a sample volume of 1 ml was used or up to 100% if urine was freeze-dried and the residue was dissolved in acetonitrile and spiked with clenbuterol prior to analysis. Chromatograms of the wash and eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of clenbuterol from calf urine.

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis.

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis.

The intra- and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra- and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra-instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8-2.6% and interinstrument reproducibilities were between 3.2-3.9%.

Fluorescent-labeled ligands for the benzodiazepine receptor. Part 2: The choice of an optimal fluorescent-labeled ligand for benzodiazepine receptor assays.

In this article, the binding affinities of the fluorescent-labeled benzodiazepines described in Part 1 are compared to assess the influence of the labeling position and the choice of fluorophore on the binding affinity. This comparison was extended by taking into account the data of other fluorescent-labeled benzodiazepines in the literature. The differences in the binding affinities observed could partly be explained by structure-activity relationships (SAR). On the basis of this comparison, fluorescent-labeled desethylflumazenil (Ro15-3890, 19) derivatives were selected as the most suitable labeled ligands in fluorescent receptor assays. A methyl-methoxycoumarin derivative (Mmc-O-CO-(CH2)3-Ro15-3890) (20b) had a Ki-value of 6.5 nM, and a 7-nitrobenz-2-oxa-1,3diazole derivative (NBD-NH-(CH2)3-Ro15-3890, 21) had a Ki-value of 5.7 nM. In order to yield sufficient sensitivity in the final receptor assay, a suitable fluorescent labeled ligand should have a Ki < 10 nM. A further advantage of the above two ligands is that the benzodiazepine moiety has no receptor affinity of its own. Thus, if some hydrolysis of the labeled ligand were to occur, the resulting Ro15-3890 (18) would hardly affect the outcome of the assay. In the second part of this paper the prerequisites of the fluorophore are being examined. In this regard, 20b is preferred, because the coumarin derivative has higher fluorescence intensities in aqueous media than the NBD-derivative. Therefore, 20b was selected as a fluorescent-labeled ligand in the development of a non-radioactive receptor assay for benzodiazepines.

Fluorescent-labeled ligands for the benzodiazepine receptor. Part 1: Synthesis and characterization of fluorescent-labeled benzodiazepines.

Because radioactive labeled ligands in receptor assays have several disadvantages, we synthesized a number of fluorescent-labeled benzodiazepines. Several fluorophores were attached at different positions of 1,4-benzodiazepine molecules in order to assess the impact of the fluorophores and their coupling position on the affinity for the benzodiazepine receptor. Besides the 1,4-benzodiazepines, the 1,2-annelated 1,4-benzodiazepines were also used for labeling. A metabolite of flumazenil (18), desethylflumazenil (Ro15-3890, 19), was labeled with the fluorophore 4-bromomethyl-7-methoxycoumarin, with and without the incorporation of a spacer chain, yielding the methyl-methoxycoumarin (Mmc) derivatives Mmc-Ro15-3890 (20a) and Mmc-O-CO-(CH2)3-Ro15-3890 (20b), respectively. After the synthesis, the fluorescent-labeled benzodiazepines were purified by HPLC, using an analytical RP-C18 column. For the purification of 20b, the chromatographic system was optimized, using multi-criteria decision making (MCDM) techniques. The binding affinities for the benzodiazepine receptor and the fluorescence characteristics were determined for the resulting products.

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine.

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

Selectivity in capillary electrokinetic separations.

This review gives a survey of selectivity modes in capillary electrophoresis separations in pharmaceutical analysis and bioanalysis. Despite the high efficiencies of these separation techniques, good selectivity is required to allow quantitation or identification of a particular analyte. Selectivity in capillary electrophoresis is defined and described for different separation mechanisms, which are divided into two major areas: (i) capillary zone electrophoresis and (ii) electrokinetic chromatography. The first area describes aqueous (with or without organic modifiers) and nonaqueous modes. The second area discusses all capillary electrophoretic separation modes in which interaction with a (pseudo)stationary phase results in a change in migration rate of the analytes. These can be divided in micellar electrokinetic chromatography and capillary electrochromatography. The latter category can range from fully packed capillaries, via open-tubular coated capillaries to the addition of microparticles with multiple or single binding sites. Furthermore, an attempt is made to differentiate between methods in which molecular recognition plays a predominant role and methods in which the selectivity depends on overall differences in physicochemical properties between the analytes. The calculation of the resolution for the different separation modes and the requirements for qualitative and quantitative analysis are discussed. It is anticipated that selectivity tuning is easier in separation modes in which molecular recognition plays a role. However, sufficient attention needs to be paid to the efficiency of the system in that it not only affects resolution but also detectability of the analyte of interest.

Detection of muscarinic receptors in the human lung using PET.

UNLABELLED: The characterization of pulmonary muscarinic receptors with PET is still in its infancy. Because approximately 70% of the lungs consists of air and pulmonary muscarinic receptor densities are low, ligands with high receptor affinity are required to obtain reasonable signal-to-noise ratios on PET images. Therefore, the potent 11C-labeled muscarinic antagonist N-methyl-piperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide ([R]-VC-002) was developed. We administered this radioligand to four healthy human volunteers to examine its suitability for studying pulmonary muscarinic receptors in vivo. METHODS: [11C]VC-002 (185 MBq, specific activity > 7.4 TBq/mmol) was intravenously injected on 2 separate days, with an interval of at least 1 wk. On the first day the volunteers were not pretreated, but on the second day they received the anticholinergic glycopyrronium bromide (Robinul; 2 x 0.1 mg intravenous) 25 and 30 min before the injection of the radiopharmaceutical. C[15O]O scans (approximately 740 MBq [20 mCi] by inhalation) were acquired before the receptor scan to calculate pulmonary blood volume. RESULTS: On PET images of the thorax, the lungs were clearly visible. After the volunteer was pretreated with glycopyrronium bromide, pulmonary uptake of the radioligand was reduced to 32%+/-12% of the control value at 60 min postinjection and the lungs could no longer be seen. (R)-[11C]-VC-002 was rapidly cleared from plasma and was slowly metabolized during the time course (60 min) of the PET scan. The fraction of radioligand representing parent compound decreased from 99.9% at the time of injection to 82% at 40-60 min postinjection, both in the presence and absence of Robinul. Pulmonary tissue-to-plasma ratios, calculated on a count-per-minute-per-gram basis, reached a plateau value of 17.8+/-1.2 at 40-50 min postinjection. CONCLUSION: [11C]VC-002 appears to be suitable for in vivo studies of pulmonary cholinoceptors.

Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis-mass spectrometry.

This paper describes the development of an analytical method for the separation and identification of neuropeptide Y (NPY) and two important NPY fragments by capillary electrophoresis (CE) and mass spectrometry (MS). A satisfactory separation and the highest sensitivity were obtained with formic acid at high concentrations (250 mM, pH 2.75). The addition of 25 or 50 mM triethylamine (TEA) improved the separation. When applying full scan CE-MS, the separated peptides could be detected and identified using the spectra of each peak. The use of TEA as an additive to the formic acid slightly decreased the sensitivity but was compensated by the improved efficiency. The best compromise for optimal separation and MS detection was found to be 50 mM formic acid to which 50 mM TEA was added. CE-MS could be used for identification of the decomposition products of NPY. Decomposition products with one amino acid difference, which could not be distinguished with CE-UV, could be distinguished with CE-MS.

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.

Capillary electrophoresis as a versatile tool for the bioanalysis of drugs--a review.

This review article presents an overview of current research on the use of capillary electrophoretic techniques for the analysis of drugs in biological matrices. The principles of capillary electrophoresis and its various separation and detection modes are briefly discussed. Sample pretreatment methods which have been used for clean-up and concentration are discussed. Finally, an extensive overview of bioanalytical applications is presented. The bioanalyses of more than 200 drugs have been summarised, including the applied sample pretreatment methods and the achieved detection limits.

Modelling of conditions for the enantiomeric separation of beta2-adrenergic sympathicomimetics by capillary electrophoresis using cyclodextrins as chiral selectors in a polyethylene glycol gel.

A two-factor central composite design was used to determine a mathematical model for prediction of the optimal conditions for the separation of the enantiomers of some widely used beta2-sympathicomimetic drugs (beta2-agonists) by capillary electrophoresis using cyclodextrins (CD) as a chiral selector in a polyethylene glycolgel. The effects of the chemical structure of these drugs along with the addition of polyethylene glycol to the cyclodextrin solution on the resolution of their enantiomers were studied. To allow impurity studies down to 0.1% (distomer eutomer) a resolution of 2.5 should be warranted. Those beta2-agonists containing two hydroxylic groups in the aromatic ring structure show the highest enantiomeric separation, due to the fact that one of their enantiomers has a better geometric structure to fit into the beta-cyclodextrin cavity.

Improved benzodiazepine radioreceptor assay using the MultiScreen Assay System.

In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time. The filtration in this method was performed by using the MultiScreen Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom, which allows both the incubation and the filtration of the specimen in the same plate. After the filtration, the filters were punched out for quantitation of the bound labeled ligand [3H]flunitrazepam. The results obtained with the MultiScreen Assay System did not differ significantly from the data obtained with the conventional filtration manifold (48S): The Ki's of lorazepam were 2.4 +/- 0.30 and 1.9 +/- 0.15 nM, respectively. In case a radioactive label is replaced by a fluorescent label, the bound labeled-ligand usually cannot be determined in the presence of the receptor material. Here, the bound labeled-ligand has to be dissociated after the filtration step. To dissociate the ligand-receptor complex, Tris- HCl buffer, containing 10 microM flumazenil, was added to the filters and the second filtrates were collected containing the previously bound fractions in the absence of receptor material. This approach showed the same Ki for lorazepam, 2.5 +/- 0.04 nM as without dissociation, when using the radio-labeled benzodiazepine [3H]flunitrazepam.