Validation of the Neogen ELISA Benzodiazepine Kit using Clonazepam as the Target Molecule for Blood and Urine.

This study demonstrates the validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using clonazepam as the target molecule for the Neogen® Benzodiazepine kit. Decision points were validated at 10.0 ng/mL for whole blood and 25.0 ng/mL for urine. The validation design was based on the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation and included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness and a case sample evaluation. The experimental limit of detection for clonazepam was determined to be at least 5.0 ng/mL in whole blood and at least 10.0 ng/mL in urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels ±50% and +100% of the decision point. Although the method was optimized and exceptional precision was demonstrated at each level, the current SWGTOX validation requirements for a valid decision point were not fulfilled. However, both the blood and urine matrix did meet the proposed revision of the SWGTOX requirements for determining a valid decision point promulgated by the American Academy of Forensic Sciences Standards Board and the assay was reliably able to detect benzodiazepines without interference from matrix components or other compounds routinely detected in authentic case samples. Case sample results were comparable with those obtained when the samples were initially screened using oxazepam as the target molecule. The Neogen® Benzodiazepine kit using clonazepam as the target molecule exhibited cross-reactivity for 29 different benzodiazepines and demonstrated excellent precision and sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for benzodiazepines, even though the validation did not fulfill current SWGTOX requirements for a valid decision point.

Detection Times of Diazepam, Clonazepam, and Alprazolam in Oral Fluid Collected From Patients Admitted to Detoxification, After High and Repeated Drug Intake.

BACKGROUND: Clonazepam, diazepam, and alprazolam are benzodiazepines with sedative, anticonvulsant, and anxiolytic effects, but their prevalence in drug abuse and drug overdoses has long been recognized. When detection times for psychoactive drugs in oral fluid are reported, they are most often based on therapeutic doses administered in clinical studies. Repeated ingestions of high doses, as seen after drug abuse, are however likely to cause positive samples for extended time periods. Findings of drugs of abuse in oral fluid collected from imprisoned persons might lead to negative sanctions, and the knowledge of detection times of these drugs is thus important to ensure correct interpretation. The aim of this study was to investigate the time window of detection for diazepam, clonazepam, and alprazolam in oral fluid from drug addicts admitted to detoxification. METHODS: Twenty-five patients with a history of heavy drug abuse admitted to a detoxification ward were included. Oral fluid was collected daily in the morning and the evening and urine samples every morning for 10 days, using the Intercept device. Whole blood samples were collected if the patient accepted. The cutoff levels in oral fluid were 1.3 ng/mL for diazepam, N-desmethyldiazepam, and 7-aminoclonazepam and 1 ng/mL for clonazepam and alprazolam. In urine, the cutoff levels for quantifications were 30 ng/mL for alprazolam, alpha-OH-alprazolam, and 7-aminoclonazepam, 135 ng/mL for N-desmethyldizepam, and 150 ng/mL for 3-OH-diazepam and for all the compounds, the cutoff for the screening analyses were 200 ng/mL. RESULTS: The maximum detection times for diazepam and N-desmethyldiazepam in oral fluid were 7 and 9 days, respectively. For clonazepam and 7-aminoclonazepam, the maximum detection times in oral fluid were 5 and 6 days, respectively. The maximum detection time for alprazolam in oral fluid was 2.5 days. New ingestions were not suspected in any of the cases, because the corresponding concentrations in urine were decreasing. Results from blood samples revealed that high doses of benzodiazepines had been ingested before admission, and explains the longer detection times in oral fluids than reported previously after intake of therapeutic doses of these drugs. CONCLUSIONS: This study has shown that oral fluid might be a viable alternative medium to urine when the abuse of benzodiazepines is suspected.

Selective extraction of clonazepam from human plasma and urine samples by molecularly imprinted polymeric beads.

A molecularly imprinted polymer (MIP) based on free-radical polymerization was prepared with 1-(N,N-biscarboxymethyl)amino-3-allylglycerol and N,N-dimethylacrylamide as functional monomers, N,N-methylene diacrylamide as the cross-linker, copper ion-clonazepam as the template and 2,2-azobis(2-methylbutyronitrile) as the initiator. The imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermo-gravimetric analysis, and SEM. The MIP of agglomerated microparticles with multipores was used for SPE. The imprinted polymer sorbent was selective for clonazepam. The optimum pH and sorption capacity were 5 and 0.18 mg/g at 20C, respectively. The profile of the drug uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. The MIP-SPE was the most feasible technique for the extraction of clonazepam with a high recovery from human plasma and urine samples.

A new chemiluminescence method for determination of clonazepam and diazepam based on 1-Ethyl-3-Methylimidazolium Ethylsulfate/copper as catalyst.

A novel chemiluminescence (CL) reaction, Benzodiazepines-H2O2-1-Ethyl-3-Methylimidazolium Ethylsulfate/copper, for determination of clonazepam and diazepam at nanogram per milliliter level in batch-type system have been described. The method relies on the catalytic effect of 1-Ethyl-3-Methylimidazolium Ethylsulfate/copper on the chemiluminescence reaction of Benzodiazepines, the oxidation of Benzodiazepines with hydrogen peroxide in natural medium. The influences of various experimental parameters such as solution pH, the ratio of 1-Ethyl-3 Methylimidazolium ethylsulfate concentration to copper ion, the type of buffer and the concentration of CL reagents were investigated. Under the optimum condition, the proposed method was satisfactorily applied for the determination of these drugs in tablets and urine without the interference of their potential impurities.

How often do false-positive phencyclidine urine screens occur with use of common medications?

BACKGROUND: Previous reports describe false-positive urine immunoassay screens for phencyclidine (PCP) associated with use of tramadol, dextromethorphan, or diphenhydramine. The likelihood of these false positives is unknown. OBJECTIVE: We sought to find the relative frequency of false-positive PCP screens associated with these medications and to look for any other medications with similar associations. METHODS: In an IRB-approved study, we retrospectively reviewed charts of all ED encounters with positive urine screens for PCP in our hospital from 2007 through 2011, inclusive. Urine samples were tested for drugs of abuse using the Siemens Syva EMIT II Immunoassay. Our laboratory routinely confirmed all positive screens using GC-MS with results classified as either "confirmed" (true positive) or "failed to confirm" (false positive). We recorded all medications mentioned in the chart as current medications or medications given before the urine sample. We used Fisher's exact test to compare frequencies of tramadol, dextromethorphan, diphenhydramine, and other medications between the two groups. RESULTS: Tramadol, dextromethorphan, alprazolam, clonazepam, and carvedilol were significantly more frequent among the false-positive group, but the latter three were also associated with polysubstance abuse. Diphenhydramine was more frequently recorded among the false-positive group, but this was not statistically significant. CONCLUSION: False-positive urine screens for PCP are associated with tramadol and dextromethorphan and may also occur with diphenhydramine. Positive PCP screens associated with alprazolam, clonazepam, and carvedilol were also associated with polysubstance abuse.

Prolonged excretion of 7-aminoclonazepam in urine after repeated ingestion of clonazepam: a case report.

Urinary analyses of the metabolite 7-aminoclonazepam (7-AC) can be helpful in monitoring drug abuse and in the context of suspected drug-facilitated sexual assaults (DFSA). Only two studies have reported detection times of 7-AC in urine after a single dose of clonazepam, and no previous studies have reported detection times after repeated ingestions of clonazepam. This report describes along detection time of 7-AC in urine in the case of a 28-year-old woman with a two year history of daily drug abuse of heroin and clonazepam, who was admitted to a detoxification unit. Urinary samples were delivered every morning for 9 days. Screening analysis in urine was performed by immunoassay, and confirmation analysis by LC-MS/MS. 7-AC was detected for 9 days, and the concentration at day 9 was still high (97 ng/ml), compared to previously reported data. These results indicate that after repeated ingestions of clonazepam, 7-AC can possibly be detected for about 2-3 weeks after cessation, applying cut-off levels commonly used in drug testing programs and DFSA cases.

Therapeutic monitoring of benzodiazepines in the management of pain: current limitations of point of care immunoassays suggest testing by mass spectrometry to assure accuracy and improve patient safety.

BACKGROUND: Concomitant use of opioids and benzodiazepines can result in significant untoward effects. Point of care (POC) urine testing devices are commonly used tools to monitor patient use of medications. These useful devices are relatively inexpensive and yield immediate results that can be acted upon at the time of the appointment, although numerous limitations have been identified for specific medications or medication classes. We established the diagnostic accuracy of a commonly used POC testing method for benzodiazepines. METHODS: One thousand patients, from a single interventional pain practice receiving opioid therapy provided urine specimens as part of the usual practice of monitoring consistency with prescribed medications. These de-identified urine specimens were tested using LC-MS/MS and the results were compared using the standard calculations for sensitivity, specificity, and predicted value. Five specimens were excluded from the study because the prescribed flurazepam could not be confirmed by LC-MS/MS (the LC-MS/MS instrumentation was not set to identify flurazepam), resulting in 995 specimens. RESULTS: Point of care assays yielded false negative results for patients prescribed benzodiazepines nearly 20% of the time (98 out of 498 patients). The point of care cup often failed to produce positive results for persons who were shown by LC-MS/MS to be taking lorazepam or clonazepam. Although only 26 out of 498 patients (5%) were prescribed ≥2 benzodiazepines, 73 out of 498 patients (15%) were found to be positive for that drug class. CONCLUSIONS: POC immunoassay for benzodiazepines could fail to provide accurate information regarding patient specific medication use. The false positive and false negative rates of the immunoassay were particularly high for clonazepam and lorazepam. Further testing of patient specimens using more accurate methods such as LC-MS/MS is necessary to provide definitive data that can assist in clinical decision making, and potentially protect these patients from untoward effects, morbidity and mortality.

Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry.

The liquid chromatography-mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and search.

Comparison of clonazepam compliance by measurement of urinary concentration by immunoassay and LC-MS/MS in pain management population.

BACKGROUND: Physicians determine patient compliance with their medications by use of urine drug testing. It is known that measurement of benzodiazepines is limited by immunoassay specificity and cutoff limits and therefore does not offer physicians an accurate picture of their patients' compliance with these medications. A few studies have used lower cutoffs to demonstrate patient compliance. OBJECTIVES: To define more appropriate cutoffs for compliance monitoring of patients prescribed clonazepam as determined using immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: A diagnostic accuracy study of the urinary excretion of clonazepam. METHODS: Millennium Laboratories performed measurements on the urinary excretion of pain patients prescribed clonazepam as the indicator test. This benzodiazepine was chosen because it forms one major metabolite, 7-aminoclonazepam which is specific for that drug. Patients whose only benzodiazepine medication was clonazepam were selected as the test population. The Millennium Laboratories test database was filtered first to select patients on clonazepam, then a second filter was used to eliminate patients with any other listed benzodiazepine medications. Samples were tested using the Microgenics DRI benzodiazepine assay with a 200 ng/mL cutoff. The same samples were quantitatively assessed for 7-aminoclonazepam by LC-MS/MS with a cutoff of 40 ng/mL. The results from the immunoassay were scored as positive or negative while the quantitative results from the LC-MS/MS were also scored as positive or negative depending upon their concentration. RESULTS: Samples from 180 patients met these medication criteria. The positivity rates were 21% (38 samples) by immunoassay. The positivity rate was 70% (126 samples) if the LC-MS/MS cutoff was set at 200 ng/mL. However, the positivity rate was 87% (157 samples) if the LC-MS/MS was set at 40 ng/mL. Concentration distributions revealed a significant fraction (7%) in the 40 - 100 ng/mL range. LIMITATIONS: A limitation of the study was the inability to measure lower than 40 ng/mL. There may be another fraction of the population that was positive below the cutoff value. CONCLUSIONS: The difference in positivity rate between the immunoassay and the LC-MS/MS result showed that the nominal 200 ng/mL cutoff of the immunoassay did not apply to 7-aminoclonazepam. This low immunoassay positivity rate is inconsistent with the manufacturer's published cross reactivity data for clonazepam and 7-aminoclonazepam. These data illustrate the limitations of using a 200 ng/mL cutoff to monitor clonazepam compliance and suggest that a cutoff of 40 ng/mL or less is needed to reliably monitor use of this drug.

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.

Ultrasensitive immunoassay of 7-aminoclonazepam in human urine based on CdTe nanoparticle bioconjugations by fabricated microfluidic chip.

The present paper described a rapid and ultrasensitive detection method using a microfluidic chip for analyzing 7-aminoclonazepam (7-ACZP) residues in human urine. A microfluidic chip-based immunoassay with laser-induced fluorescence (LIF) detection based on the water-soluble denatured bovine serum albumin (dBSA)-coated CdTe quantum dots (QDs) was prepared for the ultrasensitive detection of 7-ACZP. The whole procedure including the chip and the control software was designed and constructed in our own laboratory. The detection of 7-ACZP could be completed within 5 min. The results demonstrated that under the optima conditions, 7-ACZP residues could be detected with a precision of 5% relative standard deviation (RSD), and the linear range and the limit of detection (LOD) for 7-ACZP were 1.1-60.1 and 0.021 ngmL(-1), respectively. This method was compared with ELISA and showed a good correlation. This microfluidic chip with LIF detection was applied to the determination of 7-ACZP residues in positive human urine samples, and the results were confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). This ultrasensitive detection technique was proved to be practical for clinical use.

[Dietary supplements--surprise pills?]. / Kosttilskudd--forundringspakke i pilleform?

BACKGROUND: Sales of herbal dietary supplements have increased dramatically. A patient case drew our attention to the problem of incomplete declaration of content. METHODS: Two dietary supplements which the manufacturers claim to be natural, extremely fat-burning and energizing were analysed, as were urine and serum samples from persons taking these supplements. RESULTS: Surprisingly, the herbal dietary supplements contained drugs. Diazepam, clonazepam, ephedrine and metabolites were found when analyzing serum samples after intake of the dietary supplement Thermo-X 650, ephedrine and phenylpropanolamine after intake of the Purple Burn supplement. INTERPRETATION: Use of herbal dietary supplement can have serious consequences, for instance through interactions with drug therapy. Consumers must be given sufficient product information for safe use.

Spectrophotometric and fluorimetric determination of diazepam, bromazepam and clonazepam in pharmaceutical and urine samples.

New spectrophotometric and fluorimetric methods have been developed to determine diazepam, bromazepam and clonazepam (1,4-benzodiazepines) in pure forms, pharmaceutical preparations and biological fluid. The new methods are based on measuring absorption or emission spectra in methanolic potassium hydroxide solution. Fluorimetric methods have proved selective with low detection limits, whereas photometric methods showed relatively high detection limits. Successive applications of developed methods for drugs determination in pharmaceutical preparations and urine samples were performed. Photometric methods gave linear calibration graphs in the ranges of 2.85-28.5, 0.316-3.16, and 0.316-3.16 microgml-1 with detection limits of 1.27, 0.08 and 0.13 microgml-1 for diazepam, bromazepam and clonazepam, respectively. Corresponding average errors of 2.60, 5.26 and 3.93 and relative standard deviations (R.S.D.s) of 2.79, 2.12 and 2.83, respectively, were obtained. Fluorimetric methods gave linear calibration graphs in the ranges of 0.03-0.34, 0.03-0.32 and 0.03-0.38 microgml-1 with detection limits of 7.13, 5.67 and 16.47 ngml-1 for diazepam, bromazepam and clonazepam, respectively. Corresponding average errors of 0.29, 4.33 and 5.42 and R.S.D.s of 1.27, 1.96 and 1.14 were obtained, respectively. Statistical Students t-test and F-test have been used and satisfactory results were obtained.

Elimination of 7-aminoclonazepam in urine after a single dose of clonazepam.

The objective of this paper was to determine how long after administration of benzodiazepine clonazepam (CLO), its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in urine collected from 10 healthy volunteers who received a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for urine collection from a victim of drug-facilitated sexual assault in order to reveal drug use. A highly sensitive NCI-GC-MS method for the simultaneous quantitation of CLO and its major metabolite 7-ACLO in urine was developed and validated. The following urine samples were collected from each volunteer: one before CLO administration, and 6 h, and 1, 3, 5, 8, 10, 14, 21 and 28 days after. All urine samples (1 mL) were extracted following addition of the internal standard (D(5)-diazepam) and enzymatic hydrolysis ( beta-glucuronidase) using solid-phase extraction columns. Standard curves for CLO (500-4000 pg x mL(-1)) and 7-ACLO (50-2000 pg x mL(-1)) were prepared by spiking aliquots of negative urine. The urine from every subject was still positive for 7-ACLO 14 days after administration of the drug. Eight of the ten volunteers had measurable amounts of the metabolite 21 days after administration. One volunteer was still positive 28 days after administration. Six of the volunteers had urine concentrations of 7-ACLO that peaked at 1 day after administration. One volunteer had the highest concentration of 7-ACLO at 3 days, two volunteers at 5 days, and one at 8 days. The range of concentrations detected was from 73.0 pg x mL(-1) to 183.2 ng x mL(-1). CLO was not detected in any of the samples.

ELISA detection of clonazepam and 7-aminoclonazepam in whole blood and urine.

The ability of five commercially available enzyme-linked immunosorbent assay (ELISA) benzodiazepines to detect clonazepam and 7-aminoclonazepam in blood and urine was investigated. To determine the cross-reactivity of various ELISA assays, drug free blood and urine were fortified with clonazepam and 7-aminoflunitrazepam at concentrations of 1, 2.5, 5, 10, and 25microg/dl. The cross-reactivity, with respect to oxazepam, for clonazepam was 16, 37, 80, 93, and 109% with Immunalysis, Diagnostix, Neogen, OraSure, and Cozart, respectively; for 7-aminoclonazepam, none of the five ELISA assays showed any cross-reactivity above 10%.

Postmortem distribution and redistribution of nitrobenzodiazepines in man.

The distribution of the nitrobenzodiazepines, flunitrazepam, clonazepam and nitrazepam, and their respective 7-amino metabolites were examined in blood, serum, vitreous humor, liver, bile and urine of decedents taking these drugs. Peripheral blood, serum and liver concentrations were not significantly different to each other. However, vitreous concentrations were one-third of blood, while bile concentrations were 5-12 fold higher. Blood, serum and vitreous contained predominantly the 7-amino metabolite, liver contained only the metabolite, while bile contained significant concentrations of both the parent drug and the 7-amino metabolite. Urine contained only small concentrations of parent drug, however, as expected a number of metabolites were detected. Redistribution studies compared the drug concentrations of femoral blood, taken at body admission to the mortuary, with femoral blood taken at autopsy approximately 39 h later in 48 cases. The concentrations of 7-amino metabolites were not significantly different, however the concentrations of parent nitrobenzodiazepines were significantly higher in the admission specimens. In 6 cases in which subclavian blood was taken, the concentrations were not significantly different to the concentrations in admission blood. Similar findings were observed when femoral and subclavian blood concentrations were compared in 6 cases. There was also no apparent difference in total blood concentrations of nitrobenzodiazepines when blood concentrations taken in hospital shortly prior to death were compared to postmortem blood. Postmortem diffusion into peripheral blood is therefore not a confounding factor in the interpretation of nitrobenzodiazepine concentrations.

Identification of urinary benzodiazepines and their metabolites: comparison of automated HPLC and GC-MS after immunoassay screening of clinical specimens.

An automated high-performance liquid chromatographic method, benzodiazepines by REMEDi HS, was used to analyze benzodiazepines and their metabolites after beta-glucuronidase hydrolysis of 1-mL urine specimens from the following: 924 clinic and hospital patients whose specimens had previously been found to be presumptively positive using either EMIT or Triage immunoassay methodologies and 128 individuals whose specimens had screened negative by EMIT d.a.u.TM. REMEDi analyses did not correlate with the immunoassay results in 136 of the positive and three of the negative urine specimens. Gas chromatographic-mass spectrometric (GC-MS) confirmatory analyses were performed on these discordant specimens using 3 mL beta-glucuronidase-hydrolyzed urine followed by extraction with chloroform-isopropanol (9:1) and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. Two benzodiazepines, flunitrazepam and clonazepam, and their 7-amino metabolites were analyzed without prior derivatization. The analyses established 87% concordance between REMEDi and GC-MS versus 13% concordance with immunoassay for the subset. GC-MS analysis of these 142 specimens demonstrated two reasons for the nonconcurrence between REMEDi and EMIT: EMIT had given either false-negative or false-positive results and EMIT had given a positive result even though the determined metabolites were below the 200-ng/mL cutoff for the immunoassay and the 80-ng/mL cutoff for REMEDi. A total of 23 specimens were found to contain only lorazepam by REMEDi and GC-MS, 15 of which had been screened by Triage. A reevaluation of these 23 specimens by EMIT d.a.u. demonstrated that 11 were positive. This finding was in contrast to previous reports that EMIT will not detect lorazepam glucuronide in urine. An unexpected finding was the REMEDi identification and subsequent GC-MS confirmation of 7-aminoflunitrazepam, a urinary metabolite of flunitrazepam that is not available in the United States and that represented illicit use by four patients. A distinct advantage of REMEDi proved to be its capability in identifying demoxepam, a major metabolite of chlordiazepoxide; GC-MS analysis could not detect this metabolite because of its thermal decomposition to nordiazepam. To further evaluate the specificity of REMEDi, we conducted GC-MS analyses in a random fashion on 55 additional nondiscordant urine specimens that were identified as either positive or negative, as well as 22 specimens identified as containing 7-aminoclonazepam by REMEDi. Concurrence was observed between the two methods for all specimens, with the exception of one apparent false positive for alpha-hydroxyalprazolam by REMEDi. The reproducibility of the REMEDi method was found to be excellent; it was assessed by comparing results of 266 specimens that were reprocessed in different batches and for known calibrators and controls also processed with each batch. Study results demonstrated that the automated REMEDi assay for urinary benzodiazepines and their metabolites was comparable with GC-MS but had distinct advantages over GC-MS because of the following reasons: simplicity of the assay, less time required for analyses, and provision of additional information concerning the parent benzodiazepine.

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.

Determination of oxazepam and diazepam in body fluids by HPLC.

A reversed-phase high-performance liquid chromatography method for determination of benzodiazepine derivatives has been developed. Oxazepam and diazepam was determined in serum and oxazepam in urine.

Direct derivative spectrophotometric determination of nitrazepam and clonazepam in biological fluids.

The use of fifth order derivative spectra allows the direct determination of nitrazepam in urine at 388 nm with a limit of detection of 1 microgram ml-1. The determination of nitrazepam in blood plasma can be carried out directly by measurement at 402 nm in the fourth order derivative spectra with a limit of detection of 1.5 micrograms ml-1. Clonazepam can be determined directly in urine samples at 384 nm by using sixth order derivative spectra with a limit of detection of 1 microgram ml-1 and in blood plasma by using fourth order derivative spectra at 384 nm with a limit of detection of 0.5 ppm.