Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, -hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and -hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia β-glucuronidase for 1 h at 56°C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Direct injection micellar liquid chromatographic determination of benzodiazepines in serum

A simple micellar liquid chromatographic (MLC) procedure is reported for the determination of several benzodiazepines in serum: bromazepam, diazepam, flunitrazepam, halazepam, medazepam, nitrazepam, oxazepam and tetrazepam. The optimization studies have been made in C(18) and C(8) columns, using solutions containing sodium dodecyl sulphate (SDS) modified with butanol or pentanol as mobile phases. The method proposed for the determination of the benzodiazepines uses a hybrid micellar mobile phase of 0.06 M SDS-5% butanol-0.01 M phosphate buffer (pH 7) at 25 degrees C, and UV detection (230 nm) in a C(18) column. The serum samples were injected directly, without any pretreatment, and eluted in less than 22 min, in accordance with their relative polarities, as indicated by their octanol-water partition coefficients. The limits of detection (ng ml(-1)) were within the ranges of 2-6 and 4-18 for aqueous and serum samples, respectively. Repeatability and intermediate precision were tested for three different concentrations of the drugs, and RSD (%) was below 10 for most of the assays. The MLC results were compared with those obtained from a conventional HPLC method using methanol-water 5:5 (v/v) which requires a previous extraction procedure.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

Determination of diazepam and its pharmacologically active metabolites in blood by bond elut™ column extraction and reversed-phase high-performance liquid chromatography

A rapid and quantitative analytical micro method for the determination of diazepam and its major pharmacologically active metabolites utilizing high-performance liquid chromatography (HPLC) is reported. The drug and its metabolites were extracted from 50–100 μl samples of whole blood, serum or plasma using Bond Elut™ C_15f column and quantitated by high-performance liquid chromatography, using Technicon Fast-LC-C-8 (RP 5 μm) bonded column and a mobile phase consisting of 53% methanol, 1% acetonitrile in KH₂PO₄ buffer and 10 μl/l triethylamine. Methyl nitrazepam and medazepam were used as internal and external standards, respectively. The extraction and recovery of diazepam and its major pharmacologically active metabolites, i.e., 3-hydroxydiazepam, desmethyldiazepam and oxazepam from blood were higher than 88% for all compounds. The minimum detection range of each compound was approximately 2.5 ng per 100-μl sample. This micro method of simultaneous quantitation of diazepam and its major pharmacologically active metabolites provides a valuable technique for the study of diazepam pharmacokinetics in a small animal model without disturbance of normal hemodynamics from excess blood loss, as well as in clinical evaluation of pediatric patients.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

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.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Determination of some benzodiazepines and metabolites in serum,urine and saliva by high-performance liquid chromatography

The performance of a number of liquid—solid systems, consisting of mixtures of buffers (0.05 M) and methanol as mobile phase and methyl-silica as stationary phase, were investigated with respect to their use in the separation of 1,4-benzodiazepines by reversed-phase high-performance liquid chromatography with UV detection at 254 nm. Phase system selectivities and column efficiencies were determined. A nomogram is presented from which the chromatographic parameters can be calculated.A complete separation of nine benzodiazepines within 12 min has been achieved, using methyl-silica as the stationary phase and 50% methanol as the eluent.The results were applied to the development of a method for the determination of therapeutic levels of diazepam and its metabolites in human serum, urine and saliva. The first step in the analysis, the extraction of diazepam and its metabolites from serum and urine, was also investigated and good recoveries were achieved. A low detection limit (0.2 ng) and high precision were obtained. The concentrations of diazepam and its metabolites in human serum, urine and saliva were determined after both single and multiple oral doses of diazepam (and oxazepam).     

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.     

Rapid determination of tetracycline and lumecycline in human plasma and urine using high-performance liquid chromatography

A rapid and accurate method for the determination of tetracycline in human plasma and urine is presented. Determination of tetracycline in plasma is based on precipitation of plasma proteins with trifluoroacetic acid, followed by injection of the centrifuged plasma sample onto a μBondapak C₁₈ column. Acetonitrile in phosphate buffer pH 2.2 is used as mobile phase. Only tetracycline, and no trace of lumecycline can be detected in plasma and urine after administration of lumecycline, indicating that lumecycline is completely degraded to tetracycline, lysine and formaldehyde in the gastrointestinal tract prior to absorption.Determination of tetracycline in urine was performed by injection of urine diluted with phosphoric acid onto a μBondapak Phenyl column. The precision of determination of tetracycline in plasma, expressed as the relative standard deviation, was < 3% at tetracycline concentrations of 0.05 and 3.7 μg/ml. Urine determinations were made with a precision of < 1.5% at tetracycline concentrations of 0.5 and 6.7 μg/ml.     

Monitoring of benzodiazepines (clobazam, diazepam and their main active metabolites) in human plasma by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic method for the simultaneous determination of clobazam, diazepam and their main metabolites in human plasma is described. A 200-μl plasma sample was directly injected into a precolumn filled with TSK-gel PW. After a washing step with potassium phosphate buffer, the retained substances were backflushed into a reversed-phase column with a mobile phase of acetonitrile—phosphate buffer—diethylamine. Various drugs frequently co-administered with clobazam or diazepam do not interfere with the determination.     

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Determination of vinorelbine in rabbit plasma by high-performance liquid chromatography with coulometric detection

A high-performance liquid chromatographic method was developed for the determination in plasma (400-μl sample) of a vinca alkaloid, vinorelbine. The analysis was performed by using an octadecylsilane column and heptanesulfonic acid as ion-pairing agent. This method used a new internal standard, teniposide, that permitted a good compromise between sensitivity and retention times (10.6 and 15.5 min for teniposide and vinorelbine, respectively). After a liquid-liquid extraction with diethyl ether, the extracts were injected into a reversed-phase system. The extraction efficiency was approximately 80% for both vinorelbine and the internal standard. The mobile phase was phosphate buffer (pH 3)-acetonitrile-methanol (50:30:20, v/v/v). Using coulometric detection, the limit of detection in plasma (400 μl) was 1 ng.ml. The intra-assay coefficients of variation were 10.95, 3.80 and 5.71% for 5, 500 and 1000 ng/ml, respectively, and the inter-assay coefficients of variation were 20.14, 14.27 and 10.67% for 5, 500 and 1000 ng/ml, respectively. A linear response was observed for the plasma calibration graph in the ranges 2.5–50 and 50–1000 ng/ml. This method was used to follow the time course of the concentration of vinorelbine in rabbit plasma after a single intravenous dose of vinorelbine (30 mg/m²) and seems to be suitable for studying the pharmacokinetics of vinorelbine in rabbit.