Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Metabolism of Anxiolytics and Hypnotics: Benzodiazepines, Buspirone, Zoplicone, and Zolpidem

1. The benzodiazepines are among the most frequently prescribed of all drugs and have been used for their anxiolytic, anticonvulsant, and sedative/hypnotic properties. Since absorption rates, volumes of distribution, and elimination rates differ greatly among the benzodiazepine derivatives, each benzodiazepine has a unique plasma concentration curve. Although the time to peak plasma levels provides a rough guide, it is not equivalent to the time to clinical onset of effect. The importance of and half-lives in the actions of benzodiazepines is discussed.2. The role of cytochrome P450 isozymes in the metabolism of benzodiazepines and in potential pharmacokinetic interactions between the benzodiazepines and other coadministered drugs is discussed.3. Buspirone, an anxiolytic with minimal sedative effects, undergoes extensive metabolism, with hydroxylation and dealkylation being the major pathways. Pharmacokinetic interactions of buspirone with other coadministered drugs seem to be minimal.4. Zopiclone and zolpidem are used primarily as hypnotics. Both are extensively metabolized; N-demethylation, N-oxidation, and decarboxylation of zopiclone occur, and zolpidem undergoes oxidation of methyl groups and hydroxylation of a position on the imidazolepyridine ring system. Zopiclone has a chiral centre, and demonstrates stereoselective pharmacokinetics. Metabolic drug–drug interactions have been reported with zopiclone and erythromycin, trimipramine, and carbamazepine. Reports to date indicate minimal interactions of zolpidem with coadministered drugs; however, it has been reported to affect the C _max and clearance of chlorpromazepine and to decrease metabolism of the antiviral agent ritonavin. Since CYP3A4 has been reported to play an important role in metabolism of zolpidem, possible interactions with drugs which are substrates and/or inhibitors of that CYP isozyme should be considered.     

Simultaneous screening and quantitation of alpidem, zolpidem, buspirone and benzodiazepines by dual-channel gas chromatography using electron-capture and nitrogen—phosphorus detection after solid-phase extraction

A rapid twin-column gas chromatographic (GC) method for simultaneous screening and determination of commonly prescribed benzodiazepines and other new anxiolytics from plasma is described. Identical fused-silica Ultra 2 (5% phenyl methyl silicone) columns were connected to nitrogen—phosphorus and electron-capture detectors. The drugs were isolated from 1 ml of plasma by solid-phase extraction (SPE) onto a C₈ reversed-phase sorbent and recovered with 0.5% acetic acid in methanol. The eluate was reconstituted with isopropanol which was found suitable for on-column injection. Prazepam was used as internal standard. The method was found appropriate for the quantification in a single run of alpidem, alprazolam, buspirone, chlordiazepoxide, clobazam, clotiazepam, diazepam, estazolam, flunitrazepam, lorazepam, midazolam, oxazepam, tofisopam, triazolam, and zolpidem within 30 min. Limits of quantification allow toxicological or pharmacological determinations, except for buspirone: only toxic blood levels can be quantified by this method. This first SPE of imidazopyridines (alpidem and zolpidem) provides faster, more efficient and cheaper sample preparation than the traditional liquid—liquid procedure. This GC analysis of alpidem and zolpidem is also the first described procedure for simultaneous quantification of all different classes of anxiolytics.     

Development of a method for the determination of zopiclone in whole blood

Mass spectrometry in both electron-impact and positive chemical ionisation modes has been used to elucidate the structures of the decomposition products of zopiclone after gas chromatography. A high-performance liquid chromatographic method has been developed for the determination of zopiclone in whole blood. After selective extraction (butyl chloride) the extracts are chromatographed on Spherisorb ODS-5 (5 μm) using dibasic ammonium phosphate—acetonitrile (40:60). The zopiclone is measured by ultraviolet detection with a limit of quantitation of 4 ng/ml. This method has been successfully applied to the determination of zopiclone in post-mortem blood. Zopiclone levels found in five post-mortem cases are presented.     

Possibilities and problems with identification and determination of "new" hypnotics

Authors discuss problems with identification and determination of flunitrazepam and zolpidem in biological material (BM). Over the recent years, these two structurally different substances have become the most frequently used as well as abused hypnotic drugs. This study presents applicability of immunochemical methods in the screening of flunitrazepam, one of the most commonly prescribed drugs among the benzodiazepines. Herein described techniques, a liquid-liquid (L-L) extraction, solid phase extraction (SPE) and the so-called "freeze out" method are used for isolation of the above mentioned compounds from BM. Besides the thin layer chromatography (TLC) and gas chromatography - mass spectrometry (GC-MS) applied in qualitative analysis, the study also describes a gas chromatography with electron capture detector (GC-ECD) and gas chromatography with nitrogen phosphorus detector (GC-NPD) optimized for the determination of flunitrazepam and zolpidem in blood (serum). Successful analyses of these two substances are of major importance, especially in interpreting the results of forensic toxicological examinations.     

Determination of zopiclone in plasma using column liquid chromatography with ultraviolet detection

A reversed-phase liquid chromatographic method with ultraviolet detection for the determination of zopiclone in plasma is described. It is rapid, sensitive, reproducible and linear over a wide range. The method was used to study plasma zopiclone concentrations in a case of acute intoxication after oral ingestion of 300 mg of the drug. The plasma level was 1600 ng/ml 4.5 h after the dose and the elimination half-life was 3.5 h.     

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.     

Stereoselective binding of zopiclone to human plasma proteins

The binding of racemic zopiclone (ZOP) and of its two enantiomers to plasma proteins, albumin and α1‐acid glycoprotein were compared. Our work shows that the binding of ZOP to human plasma proteins is stereoselective. The total plasma protein binding percentages were 79.3 ± 5.5%, 83.8 ± 5.2%, and 75.1 ± 2.1%, for racemic zopiclone, (−)zopiclone and (+)zopiclone, respectively. These results were confirmed by the analysis of samples obtained from healthy volunteers after the oral administration of ZOP. The anticoagulant used for sampling was also shown to have an influence on the percentage binding and on its stereoselectivity. Considering albumin and α1‐acid glycoprotein separately, stereoselectivity was also observed. Chirality 11:129–132, 1999. © 1999 Wiley‐Liss, Inc.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

Gas chromatographic determination of zopiclone in plasma after solid-phase extraction

A gas chromatographic technique for determining zopiclone based on a solid-phase extraction procedure with C₁₈ cartridge for sample clean-up is presented. Quantification can be achieved with 1 ml of plasma. The method uses prazepam as internal standard. Zopiclone is separated on a 5% phenyl methyl silicone analytical column and detected with an electron-capture detector, which consequently allows a limit of quantitation of 2 μg/l. It is thus simple, rapid, sensitive and linear over the range 5–2000 μg/l.     

HPLC-ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.     

Acute effects of zopiclone on blood glucose level and serum lipids in hyperlipidemic rats. Interactions with PK 11195 and flumazenil

Intraperitoneal administration of 5 mg/kg zopiclone a cyclopyrolone acting on the central benzodiazepine receptors was found to produce significant reduction of total lipids, total cholesterol and triglyceride in rats randered hyperlipidemic by intraperitoneal injection of Triton W-1339. Blood glucose level was also reduced. Flumazenil (10 mg/kg) potentiated the hypoglicemic effect of zopiclone but had no additional effect on serum lipids. PK 11195 (25 mg/kg) antagonized the hypolipidemic effects of zopiclone. In conclusion: 1. The central benzodiazepine receptors are not involved in the hypolipidemic activity of zopiclone. 2. The peripheral type benzodiazepine receptors are partly responsible, for the hypolipidemic activity of this cyclopirrolone. 3. The changes of blood glucose level induced by these drugs does not seem to be related to benzodiazepine receptors.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Determination of zopiclone enantiomers in plasma by liquid chromatography using a chiral cellulose carbamate column

The enantiomers of zopiclone were determined in human plasma using a sequential achiral—chiral liquid chromatographic method. Zopiclone was separated from the biological matrix and quantified on an achiral silica column. The limit of detection was 5 ng/ml. The eluent fraction containing zopiclone was collected, evaporated, reconstituted with the mobile phase and injected onto a chiral cellulose carbamate column where the enantiomeric ratio was calculated. This validated method, applied to a pilot study, suggests that pharmacokinetics of zopiclone is stereoselective.     

生物检材中佐匹克隆的GC/μECD测定

目的：建立液-液萃取、GC/μECD气相色谱测定生物检材中佐匹克隆的方法。方法：以家兔为模型动物,500 mg佐匹克隆标准品灌胃家兔,6.5小时后处死,取其胃、肝、血、脑、心脏、尿,采用乙醚液-液萃取提取,Rtx-5毛细管柱分离,电子捕获检测器检测,标准曲线法定量。结果：该方法灵敏度达0.02μg/mL,在0.05-50μg/mL范围内线性关系良好;低、中、高3种浓度的加样回收率分别为91.7%、99.5%、94.4%,相对标准偏差（RSD）均小于5%;佐匹克隆在家兔脏器中的浓度分别为7.31μg/mL（血液）、7.63μg/g（肝脏）、7.96μg/g（脑）、14.67μg/g（心脏）、32.18μg/g（胃组织）、42.65μg/mL（尿液）,在各脏器中呈特异性分布。结论：该法操作简单、灵敏度高,结果可靠,完全满足临床检测及公安机关在办理佐匹克隆麻醉案件中的需要。     

The Stabilization of Amorphous Zopiclone in an Amorphous Solid Dispersion

Zopiclone is a poorly soluble psychotherapeutic agent. The aim of this study was to prepare and characterize an amorphous form of zopiclone as well as the characterization and performance of a stable amorphous solid dispersion. The amorphous form was prepared by the well-known method of quench-cooling of the melt. The solid dispersion was prepared by a solvent evaporation method of zopiclone, polyvinylpyrrolidone-25 (PVP-25), and methanol, followed by freeze-drying. The physico-chemical properties and stability of amorphous zopiclone and the solid dispersion was studied using differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), hot-stage microscopy (HSM), X-ray diffractometry (XRD), solubility, and dissolution studies. The zopiclone amorphous solid-state form was determined to be a fragile glass; it was concluded that the stability of the amorphous form is influenced by both temperature and water. Exposure of amorphous zopiclone to moisture results in rapid transformation of the amorphous form to the crystalline dihydrated form. In comparison, the amorphous solid dispersion proved to be more stable with increased aqueous solubility.KEY WORDS: amorphous, fragile, solid dispersion, stability, zopiclone     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Involvement of the K+‐Cl− co‐transporter KCC2 in the sensitization to morphine‐induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti‐anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K⁺‐Cl^? co‐transporter 2 (KCC2) in the sensitization to morphine‐induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ‐aminobutyric acid A‐type receptor (GABA_AR) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine‐induced hyperlocomotion, which is accompanied by the up‐regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down‐regulation of protein phosphatase‐1 (PP‐1) as well as the up‐regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP‐1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre‐treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine‐induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ‐PP‐1‐KCC2 pathway by chronic treatment with zolpidem.