Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography-tandem mass spectrometry.

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 µL) mixed with 80 µL of the IS solution were centrifuged. An aliquot (5 µL) of the sample solution was directly injected into the LC-MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 µm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5-20 ng/mL (zolpidem), 1.0-40 ng/mL (flurazepam and temazepam), 2.5-100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0-200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10-400 ng/mL (lorazepam and desalkylflurazepam) and 10-100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r(2)) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and -12.8 to 12.5%, respectively. The process efficiency for this method was 63.0-104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than -10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

LC-MS/MS analysis of 13 benzodiazepines and metabolites in urine, serum, plasma, and meconium.

We describe a single method for the detection and quantitation of 13 commonly prescribed benzodiazepines and metabolites: alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam and 7-aminoclonazepam in urine, serum, plasma, and meconium. The urine and meconium specimens undergo enzyme hydrolysis to convert the compounds of interest to their free form. All specimens are prepared for analysis using solid-phase extraction (SPE), analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and quantified using a three-point calibration curve. Deuterated analogs of all 13 analytes are included as internal standards. The instrument is operated in multiple reaction-monitoring (MRM) mode with an electrospray ionization (ESI) source in positive ionization mode. Urine and meconium specimens have matrix-matched calibrators and controls. The serum and plasma specimens are quantified using the urine calibrators but employing plasma-based controls. Oxazepam glucuronide is used as a hydrolysis control.

Determination of small drug molecules by capillary electrophoresis-atmospheric pressure ionization mass spectrometry.

Capillary electrophoresis (CE) separations are reported for sulfonamides and benzodiazepines in an uncoated fused-silica capillary. The capillary column exit was connected to a liquid junction-ion spray interface combination coupled to an atmospheric pressure ionization (API) triple quadrupole mass spectrometric (MS) system. On-line UV detection occurred 20 cm from the inlet of the capillary and with the API mass spectrometer (CE-API-MS) after the entire length of the capillary (100 cm). The separations were made using volatile buffers composed of ammonium acetate (15-20 mM) with 15-20% of methanol to facilitate ionization under electrospray conditions. This study showed that the major metabolite of flurazepam in man, N-1-hydroxyethylflurazepam, could be detected and characterized in human urine by CE-UV-MS following the administration of a single oral dose of 30 mg of flurazepam dihydrochloride. The presence of additional flurazepam metabolites in human urine was observed by using the system, suggesting that a combination of UV with MS detection should be useful for metabolic studies. In addition to molecular weight determination of compounds, structural information may be obtained by utilizing online tandem mass spectrometry (CE-UV-MS-MS). This was demonstrated for sulfamethazine where the protonated molecule species was transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of the protonated sulfamethazine molecule yielded structural information characteristic of the sulfa drug following the on-column injection of 2 pmol of sulfamethazine.

Direct liquid inlet liquid chromatographic/mass spectrometric identification and high-performance liquid chromatographic analysis of a benzodiazepine glucuronide.

The glucuronide of N-1-hydroxy-ethyl flurazepam has been analysed by a direct liquid inlet liquid chromatographic/mass spectrometric system using MeOH/H2O (70:30 v/v) as mobile phase, at a flow rate of 0.7 ml min-1. Urine samples were purified by amberlite XAD-2 chromatography; the glucuronide was quantified by high-performance liquid chromatography using a counterion (tetrabutyl ammonium nitrate in methanol). Chromatographic results were validated by an enzymatic method: treatment of the samples with beta-glucuronidase and extraction of the parent drug with ethyl ether at pH 9. The biological application of this method was demonstrated by determination of this glucuronide in the urine of healthy human volunteers following a single intravenous administration of 50 mg of N-1-hydroxy-ethyl flurazepam.

Determination of the major urinary metabolite of flurazepam in man by high-performance liquid chromatography.

A high performance liquid chromatographic method for the determination of N-1-hydroxyethylflurazepam, the major urinary metabolite of flurazepam, in human urine is described. Urine specimens were incubated enzymatically to deconjugate N-1-hydroxyethylflurazepam glucuronide (metabolite) and were then extracted at pH 9.0 to extract the metabolite. The extracts were chromatographed on a microparticulate silica gel column using automatic sample injection, isocratic elution at ambient temperature and UV monitoring at 254 nm. The internal standard was 7 chloro-5(2'-chlorophenyl) 1,3-dihydro-1-2-dimethylaminoethyl-2H-1,4-benzodiazepine-2-one. The recovery from urine, in the 0.5-25.0 microgram/ml range, was 96.5 +/- 11.5% (S.D.), and the sensitivity limit was 0.5 microgram/ml. The method was found to be specific for N-1-hydroxyethylflurazepam in the presence of intact flurazepam and other possible urinary metabolites of flurazepam. The method was successfully applied to urine specimens collected from human subjects following the administration of 30-mg single oral doses of flurazepam dihydrochloride.

GLC-ECD determination of 1-(2-hydroxyethyl)-3-hydroxy-7-chloro-1,3-dihydro-5-(O-fluorophenyl)-2H-1,4-benzodiazepin-2-one (SAS 643) in plasma and urine and identification of its main biotransformation products.

A method using gas-liquid chromatography-electron-capture detection method is described for rapid, accurate determination of SAS 643 in plasma and urine. The drug is extracted from biological fluid with benzene and converted to the O, O'-bistrimethylsilyl derivative with bis(trimethylsilyl) trifluoroacetamide. The glucuronide form of the drug is extracted after hydrolysis with beta-glucuronidase. Nimetazepam is used as internal standard. Moreover, some metabolites such as glucuronide and the N-1-dealkylated and N-1-yl-acetic products are identified. All compounds were confirmed by thin-layer chromatography, mass spectroscopy, and gas-liquid chromatography-mass spectroscopy by comparison with reference products.

The gas chromatography mass spectrometry of the major metabolites of flurazepam.

Mass spectra and gas chromatographic data are presented for flurazepam and its metabolites; monodesethylflurazepam, didesethylflurazepam, hydroxyethylflurazepam, N1-desalkylflurazepam, N1-desalkyl-3-hydroxy-flurazepam, and flurazepam-N1-acetic acid. The on-column thermal degradation of didesethylflurazepam, N1-desalkyl-3-hydroxyflurazepam and flurazepam-N1-acetic acid is reported and discussed. Mass spectrometric and gas chromatographic data are also presented for the benzophenones obtained by acid hydrolysis of flurazepam and its metabolites. The occurrence of flurazepam metabolites in urine from five forensic cases after various treatments has been investigated. A possible new 'metabolite' of flurazepam was detected in two of these cases.

Identification of flurazepam (Dalmane) and a primary metabolite in urine by thin-layer chromatography.

A relatively simple, routine method has been described for the qualitative identification of flurazepam and its primary metabolite 7-chloro-1-(2-hydroxyethyl)-5-(2-fluorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one in urine. We have described two extractions and several identification procedures by which flurazepam and its primary urinary metabolite can be identified by TLC.