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.

[Cross-reactivity of Instant-View M-1 for detection of benzodiazepine-related drugs and their metabolites in urine].

Immunoassays are useful methods for the determination of regulated drugs in clinical and forensic laboratories. Although the Instant-View M-1 (IV M-1) immunoassay kit is frequently used to screen drugs in laboratories in Japan, basic information about the IV M-1 such as its specificity and reactivity is not available. In this study, we determined the specificity and cross-reactivity of IV M-1 for the detection of benzodiazepine-related drugs and their metabolites in urine. The IV M-1 could detect triazolobenzodiazepines such as triazolam in urine at concentrations > or = 300 ng/mL. However, thienodiazepines such as etizolam could not be detected because of lack of cross reactivity. A correlation was observed between the structure of the metabolites and the reactivity of the kit; 4-hydroxy metabolites of alprazolam and triazolam were detectable, whereas a-hydroxy metabolites were not. Furthermore, 7-amino metabolites such as nitrazepam could not be detected at any concentration, including high concentrations. The specificity and reactivity of various kits used for detection of drugs in urine are different. Therefore, it is necessary to consider the basic features of the kit used while assessing the results obtained.

Analysis of quazepam and its metabolites in human urine by gas chromatography-mass spectrometry: application to a forensic case.

A sensitive method for the simultaneous determination of quazepam and two of its metabolites, 2-oxoquazepam and 3-hydroxy-2-oxoquazepam, in human urine was developed using gas chromatography-mass spectrometry (GC/MS) with an Rtx-5MS capillary column. The quazepam and its metabolites were extracted from human urine using a simple solid-phase extraction Oasis(®) HLB cartridge column, and the 3-hydroxy-2-oxoquazepam was derivatised using BSTFA/1%TMCS and pyridine at 60 °C for 30 min. The mass spectrometric detection of the analytes was performed in the full scan mode, m/z 60-480, and selected ion monitoring (SIM) mode, m/z 386, for quazepam; m/z 342, for 2-oxoquazepam; m/z 429, for 3-hydroxy-2-oxoquazepam-TMS; and m/z 284, for alprazolam-d5 (internal standard), by electron ionization. The calibration curves of quazepam and its metabolites in urine showed good linearity in the concentration range of 2.5-500 ng/0.2 ml of urine. The average recoveries of quazepam and its metabolites from 0.2 ml of urine containing 500 ng and 50 ng of each drug were 71-83% and 88-90%, respectively. The limits of detection of quazepam, 2-oxoquazepam and 3-hydroxy-2-quazepam in urine by the selected ion monitoring mode were 0.096-0.37 ng/ml. This method would be applicable to other forensic biological materials containing low concentrations of quazepam and its metabolites.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.

A drug rape case involving triazolam detected in hair and urine.

In recent years, there has been heightened awareness regarding the use of drugs to modify a person's behavior to facilitate crime. A drug rape case involving the potent, short-acting sedative triazolam will be presented. On three occasions, the victim consumed green tea and chocolate before being massaged and ultimately sexually abused. Screening for alcohol, commonly used drugs and illicit substances in blood and urine sampled during the forensic examination 20 h after the last incident, was negative. Consequently, hair samples for chemical analysis were taken from the assaulted individual 34 days after the last incidents. The hair was cut into three 2-cm segments (0-6 cm) that were washed, dissolved in extraction solvent and screened and verified by ultra performance liquid chromatography coupled with time of flight mass spectrometry (UPLC-TOF-MS) and with tandem mass spectrometry (UPLC-MS/MS), respectively. In the 2-cm hair segment corresponding to the period of the alleged assaults, the presence of the sedative triazolam was revealed at a concentration of 1.0 pg/mg hair. The preserved urine sample, taken 20 h after the last incident, was reanalyzed by UPLC-MS/MS for metabolites of triazolam, and 39 µg/l α-hydroxytriazolam was detected in the hydrolyzed urine. This case illustrates that hair is a valuable forensic specimen in situations where natural processes have eliminated the drug from typical biological specimens due to delays in the crime being reported. Furthermore, it was possible to verify the hair finding with a urine sample by detection of a metabolite of triazolam.

Imprinted functionalized silica sol-gel for solid-phase extraction of triazolamin.

A triazolam-imprinted silica microsphere was prepared by combining a surface molecular-imprinting technique with the sol-gel process. The results illustrate that the triazolam-imprinted silica microspheres provided using γ-aminopropyltriethoxysilane and phenyltrimethoxysilane as monomers exhibited higher selectivity than those provided from γ-aminopropyltriethoxysilane and methyltriethoxysilane. In addition, the optimum affinity occurred when the molar ratio of γ-aminopropyltriethoxysilane, phenyltrimethoxysilane, and the template molecule was 4.2:4.7:0.6. Retention factor (k) and imprinting factor (IF) of triazolam on the imprinted and non-imprinted silica microsphere columns were characterized using high performance liquid chromatography (HPLC) with different mobile phases including methanol, acetonitrile, and water solutions. The molecular selectivity of the imprinted silica microspheres was also evaluated for triazolam and its analogue compounds in various mobile phases. The better results indicated that k and IF of triazolam on the imprinted silica microsphere column were 2.1 and 35, respectively, when using methanol/water (1/1, v/v) as the mobile phase. Finally, the imprinted silica was applied as a sorbent in solid-phase extraction (SPE), to selectively extract triazolam and its metabolite, α-hydroxytriazolam, from human urine samples. The limits of detection (LOD) for triazolam and α-hydroxytriazolam in urine samples were 30 ± 0.21 ng mL(-1) and 33 ± 0.26 ng mL(-1), respectively.

Simultaneous quantification of triazolam and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

A simple, simultaneous, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of triazolam and its metabolites, α-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), in human urine was developed and validated. Triazolam-d4 was used as the internal standard (IS). This analysis was carried out on a Thermo(®) C(18) column, and the mobile phase was composed of acetonitrile/H(2)O/formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem MS using positive ion mode electrospray ionization, and quantification was performed by multiple reaction monitoring mode. The MS-MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 331.0, 359.0 → 111.2, and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ, and triazolam-d(4), respectively. The lower limits of quantification of the analytical method were 0.5 ng/mL for triazolam, 5 ng/mL for α-OHTRZ, and 0.5 ng/mL for 4-OHTRZ. The within- and between-run precisions were less than 15%, and accuracy was -12.33% to 9.76%. The method was proved to be accurate and specific, and it was applied to a urinary excretion study of triazolam in healthy Chinese volunteers.

Quantitation of benzodiazepines in blood and urine using gas chromatography-mass spectrometry (GC-MS).

The benzodiazepine assay utilizes gas chromatography-mass spectrometry (GC-MS) for the analysis of diazepam, nordiazepam, oxazepam, temazepam, lorazepam, alpha-hydroxyalprazolam, and alpha-hydroxytriazolam in blood and urine. A separate assay is employed for the analysis of alprazolam. Prior to solid phase extraction, urine specimens are subjected to enzyme hydrolysis. The specimens are fortified with deuterated internal standard and a five-point calibration curve is constructed. Specimens are extracted by mixed-mode solid phase extraction. The benzodiazepine extracts are derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSFTA) producing tert-butyldimethyl silyl derivatives; the alprazolam extracts are reconstituted in methanol without derivatization. The final extracts are then analyzed using selected ion monitoring GC-MS.

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 triazolam in human urine by gas chromatograph-micro electron capture detector].

Triazolam is often abused in criminal activities. It is necessary to develop a simple, fast and sensitive method for determining triazolam in human urine. After the triazolam in urine was extracted with ether at pH 9, the analysis was performed on an HP-5 capillary column with gas chromatograph-micro electron capture detector (GC-muECD). The calibration curve of triazolam showed good linearity in the range of 0.2 - 50 ng/mL with the correlation coefficient of 0.999 5. The inter-day relative standard deviation (RSD) and intra-day RSD were 4.17% and 5.31%, respectively. The average recovery was 93.9%. This simple, sensitive and accurate method can be easily implemented for monitoring triazolam in urine.

Urinary excretion profiles of two major triazolam metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam.

The objective of this study was to examine urinary excretion profiles of two major triazolam metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ) in humans. Urine samples were collected from three healthy male volunteers who had been previously administered single 0.25- and 0.5-mg doses of triazolam 24 h and 48 h, respectively, before sample collection. After enzymatic hydrolysis and extraction, each sample was analyzed by liquid chromatography-mass spectrometry. alpha-OHTRZ was rapidly excreted, with the maximum concentrations appearing in the first or second sample collected after ingestion, with the majority of the drug being excreted within 12 h. Meanwhile, 4-OHTRZ was excreted more slowly than alpha-OHTRZ. The alpha-OHTRZ/4-OHTRZ ratios were initially greater than 19.7, then decreased rapidly, reaching a nearly constant value for times in excess of 12 h.

Urinary excretion of alpha-hydroxytriazolam following a single dose of halcion.

As an approved medicinal product and reportedly an abused substance that have been associated with death and "considered to be a factor...of impaired driving, sexual assault, and other violent crimes", triazolam is controlled at the same level (Level III) as flunitrazepam in Taiwan. Alleged misuses of this substance have been associated with case specimens submitted to this laboratory. A sample preparation (with and without enzymatic hydrolysis) and gas chromatography-mass spectrometry protocols were evaluated and applied to the analysis of free and total alpha-hydroxytriazolam (the main metabolite of triazolam) in urine. Ions designated for TMS-derivatized alpha-hydroxytriazolam and alpha-hydroxytriazolam-d4 are m/z 415, 417, and 430 and 419, 421, and 434, respectively. The overall protocol achieved the following results when applied to the analysis of 2-mL drug-free urine specimens fortified with 10-200 ng/mL alpha-hydroxytriazolam: recovery, 95%; interday and intraday precision ranges, 1.50-3.52% and 0.93-4.71%, respectively; linearity, r2 > 0.99; and limits of detection and quantitation, 0.05 and 0.1 ng/mL, respectively. This protocol was applied to the analysis of case specimens and urine samples collected from two patients (A and B) taking one oral dose of Halcion (0.25 mg triazolam). Excretion profiles of free and total alpha-hydroxytriazolam show that free alpha-hydroxytriazolam is detectable, but at very low levels (< 5 ng/mL). Peak excretion of total alpha-hydroxytriazolam occurs at approximately 5-10 h following the drug intake. Total alpha-hydroxytriazolam is excreted at detectable levels approximately 2-35 h following an oral dose of 0.25 mg triazolam. Total free and conjugated alpha-hydroxytriazolam excreted by A and B are 0.61% and 31.6%; and 0.36% and 57.2% of the dose, respectively.

[Identification of estazolam, alprazolam and triazolam in human urine by LC/MSn].

AIM: To investigate the fragmentation behavior of triazolobenzodiazepines and to develop a specific, sensitive and rapid LC/MSn assay for simultaneous determination of estazolam, alprazolam and triazolam in human urine. METHODS: After oral administration of a single 4 mg dose of the drugs to each of three healthy volunteers, urine samples were purified by solid-phase extraction, and then injected into an ODS column (150 mm x 4.6 mm) with a mobile phase of methanol-water (8:2) for LC/MSn analysis. The structures of estazolam, alprazolam and triazolam in human urine were identified by direct comparison of the observed mass spectra and the chromatographic retention time with those of the reference substance. The mass spectrometer (Finnigan LCQ) was operated in positive mode and in two scan modes including SIM and full scan MS/MS mode. The obtained mass spectra was analyzed assisted with the software Mass Frontier 1.0 for their fragmentation pathways. RESULTS: The full scan MS/MS spectra of each compound gave characteristic fragment ions of [M + H - N2]+ and [M + H - Cl]+. The detection limit was below 0.5 ng.mL-1 for estazolam, alprazolam and triazolam in human urine. CONCLUSION: The method is useful in forensic and clinical toxicology in which unequivocal identification of eatazolam, alprazolam and triazolam is desired.

Improved cross-reactivity to alpha OH triazolam in the BMC CEDIA DAU urine benzodiazepine assay.

Immunoassays designed to detect use of older benzodiazepines such as oxazepam or diazepam often cannot detect triazolam use because of the low doses of triazolam administered, rapid biotransformation to metabolites with poor cross-reactivities, and the small amount of alpha OH triazolam glucuronide excreted in the urine. Previous studies have demonstrated that certain immunoassays have high cross-reactivity to alpha OH triazolam but are unable to detect therapeutic triazolam use in urine. The objectives of this study were to characterize the immunoreactivity toward alpha OH triazolam in the reformulated cloned enzyme donor immunoassay (CEDIA) drug abuse urine benzodiazepine assay and to measure the immunoreactivity of urine specimens from subjects who were administered single oral doses of triazolam. Alpha OH triazolam standards were prepared in drug-free urine and the new CEDIA assay gave a positive result at concentrations from 100 to 200 ng/ml, which indicates an eight-fold improvement in CEDIA cross-reactivity to alpha OH triazolam standards in the reformulated CEDIA assay. With a 200 ng/ml cut-off, 4/30 of the urine specimens screened positive for benzodiazepines without enzymatic hydrolysis and 6/30 after enzymatic hydrolysis. When using an in-house 100 ng/ml nitrazepam cut-off calibrator, 10/30 urine specimens were positive in the reformulated CEDIA assay without hydrolysis and 22/30 were positive with enzymatic hydrolysis before screening.

[Determination of triazolam in human urine by GC/ECD].

A GC/ECD method was established to determine triazolam in human urine qualitatively and quantitatively. The triazolam was extracted from 2 ml urine with 2 x 2 ml chloroform. The retention time of triazolam was 10.74 min. The limit of the determination was 0.5 ng/ml. The recovery of the procedure in 5 ng/ml was 95.98%, CV = 7.85% (n = 5). The limit of quantitation is 2-50 ng/ml.

Comparative evaluation of five immunoassays for the analysis of alprazolam and triazolam metabolites in urine: effect of lowering the screening and GC-MS cut-off values.

This study included evaluation of five commercially available immunoassays for the detection of alprazolam and triazolam metabolites in urine following single oral doses of these drugs. The products investigated were the EMIT d.a.u. assay, EMIT II assay, Abbott TDx (FPIA) assay, Bio Site TRIAGE device, and the Boehringer Mannheim/Microgenics CEDIA assay for urinary benzodiazepines. Urine specimens were also analyzed quantitatively by gas chromatography-mass spectrometry. Percent cross-reactivity was assessed by analysis of drug free urine containing drug standards at concentrations ranging from 100 to 10,000 ng/mL. The drug standards analyzed were alpha-OH-alprazolam, alpha-OH-triazolam, and alpha-OH-alprazolam glucuronide. The effect of lowering the screening cut-off value to 100 ng/mL, lowering the confirmation cut-off value to 50 and 25 ng/mL and the use of beta-glucuronidase hydrolysis prior to analysis was also studied. Lowering the screening cut-off value and using enzymatic hydrolysis prior to screening increased the positive detection rate for benzodiazepines with the EMIT d.a.u. assay and fluorescence polarization immunoassay (FPIA). The TRIAGE device gave the lowest percent cross-reactivity in the analysis of the drug standards and gave negative results in all urine specimens analyzed following ingestion of alprazolam and triazolam.

Identification of parent benzodiazepines by gas chromotography/mass spectroscopy (GC/MS) from urinary extracts treated with B-glucuronidase.

Urinary glucuronide metabolites of the benzodiazepines were converted back to the parent molecules after treatment with B-glucuronidase. The benzodiazepines were extracted by a one-step liquid/liquid extraction from urine or by a liquid/solid phase extraction. For the limit of detection (LOD), a standard solution of diazepam and oxazepam was serially diluted and analyzed to the point at which a reproducible analytical result was no longer obtained. Using a temperature program and a splitless mode of injection, excellent quantitation was achieved within an 8-min run time. Based upon specimens obtained from patients under a physician's care, we have determined that urinary concentrations of the benzodiazepines > 200 ng/ml are most likely due to abuse rather than to a prescribed ingestion under strict medical surveillance. Therefore, the calibration standard and cutoff concentration for a positive result was set at 200 ng/ml.