Benzodiazepines In Urine Research Articles

Gas chromatographic–mass spectrometric confirmation of selected benzophenones from benzodiazepines in human urine following automatic screening

This paper reports a simple, expeditious procedure for confirming the presence of benzodiazepines in previously screened positive urine samples. Samples are manually hydrolysed to the corresponding benzophenones in an acid medium. The hydrolysis products are continuously aspirated into a photometric flow system for screening; positive samples are then confirmed and benzodiazepines identified by using a different flow system that conditions the analytes for gas chromatographic separation and unequivocal mass spectrometric confirmation. Detection limits for benzodiazepines in the nanogram-per-millilitre region are thus achieved by using 0.5 ml of hydrolysed urine; also, repeatability, as R.S.D., is less than 6.5%. The high specificity and sensitivity of the proposed method enables the confirmation of different benzodiazepines in urine samples obtained from drug addicts and patients from a local hospital.

Solid phase micro extraction coupled with semi-microcolumn high performance liquid chromatography for the analysis of benzodiazepines in human urine

SPME/semi-microcolumn HPLC (SPME/LC) was investigated to analyze benzodiazepines in human urine samples. SPME conditions such as extraction time, extraction temperature, salt concentration and pH of matrix, flush volume and desorption time were optimized by extracting various drugs from a prepared water matrix. Combination of adding saturated salts to the matrix and controlling pH ranged from neutral to weakly alkaline conditions makes the increase of extraction efficiency. Under optimal condition SPME/LC is more sensitive than direct HPLC analysis without the SPME process. The limits of detection (LODs) was several ppb level and the relative standard deviation (RSD) was <15% when human urine samples were analyzed by this analytical system. The system is very useful and is enough to assay benzodiazepines in a human urine sample without tedious and complex analytical procedures. In this paper the applicability of SPME/LC to the analysis of benzodiazepines in human urine samples was reported. In addition, the extension to the evaluation of SPME/LC/MS system was also described.

CEDIA dau Benzodiazepine screening assay: a reformulation.

The CEDIA dau Benzodiazepine assay has been reformulated to include online hydrolysis of urinary benzodiazepine glucuronide conjugates. The new antibody possesses enhanced cross-reactivities toward the low-dose benzodiazepines, which are excreted at low urinary drug-metabolite concentrations. The screening method was evaluated using lorazepam as the probe benzodiazepine. Four subjects each consumed a 1-mg lorazepam tablet. Sequential urine voids over the same time intervals were collected for the next 48 h. Twelve postdose urine samples were collected from each subject. Positive results were obtained from 5-24 h to 2-35 h using a 200-ng/mL nitrazepam calibration cutoff. There was no practical difference between hydrolyzing online with the supplied E. coli beta-glucuronidase or offline with Helix pomatia beta-glucuronidase purchased separately. Without hydrolysis, all urine samples tested negative. The cross-reactivities of lorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% for lorazepam concentrations between 50 and 2500 ng/mL. Lorazepam glucuronide gave cross-reactivities (expressed as lorazepam base) between 72 and 136% using the online hydrolysis procedure with E. coli beta-glucuronidase. Offline hydrolysis with Helix pomatia gave cross-reactivities between 84 and 134%. Without hydrolysis, lorazepam glucuronide gave less than 4% cross-reactivity in the assay.

An automated screening system for benzodiazepines in human urine

A rapid flow system for determining the presence or absence of benzodiazepines in human urine has been developed and optimized. The system is based on the diazotization of the analytes in a pre-hydrolyzed samples at pH<1, followed by the formation of dyes with 1-naphthol at pH>12. The dyes are retained and washed on a XAD-2 minicolumn, and immediately eluted with 3 M NaOH in ethanol/water. A simple photometer is used at λ=550 or 600 nm to monitor the overall analyte content. The reliability of the proposed method for the benzodiazepines is demonstrated in simple chemometric terms, using four concentration levels (between 0.5 and 2 times the detection limit) and oxazepam as model benzodiazepine. The method is highly selective and a sensitive enough for detecting benzodiazepines in urine.

Microcolumn liquid chromatography coupled with solid phase micro extraction (SPME/micro-LC) for the analysis of benzodiazepines in human urine

Microcolumn liquid chromatography coupled with solid phase micro extraction (SPME/micro-LC) for the analysis of benzodiazepines in human urine

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.

Comparison between the CEDIA and EMIT II immunoassays for the determination of benzodiazepines

We evaluated a new, qualitative immunoassay for benzodiazepines in urine using CEDIA technology on the Hitachi 747 and compared its performance to an immunoassay using EMIT II methodology on the same instrument. A total of 500 urine samples received for routine drug screen analysis were prospectively examined for benzodiazepines by both methods. Samples producing positive results by either immunoassay method were analyzed by gas chromatography–mass spectrometry (GC–MS). Available medical records were reviewed for patients whose samples produced discrepant immunoassay results or that were positive in both immunoassays but negative by GC–MS. Samples that produced negative results in both immunoassays were not subjected to GC–MS analysis. Therefore, identification of an immunoassay result as a false negative only occurred when the sample produced a positive value in only one of the two immunoassays and was confirmed as positive by either GC–MS or medical record review. Following initial immunoassay screening and confirmation by GC–MS, a medical record review and reanalysis of GC–MS data was performed. After this in-depth analysis of the data, the CEDIA method produced 60 true-positives, 7 false positives and no false negatives. The EMIT II method produced 47 true positives, 1 false positive, and 13 false negatives. These differences appear to be due to the CEDIA assay being more sensitive for detection of lorazepam.

Micro-extraction of commonly abused benzodiazepines for urinary screening by liquid chromatography.

We have developed a micro-extraction procedure for the analysis of seven commonly prescribed benzodiazepines (chlordiazepoxide, diazepam, lorazepam, nitrazepam, nordiazepam, oxazepam, and temazepam) in urine using liquid chromatography. The method is reliable and sensitive, uses small volumes (100 microL) of urine and is suitable for the detection and quantification of low concentrations of benzodiazepines. The micro-extraction procedure allowed rapid sample processing, which is important for routine sample handling. The limit of detection for the seven benzodiazepines ranged from 0.10-0.71 mg/L and recovery of the different benzodiazepines was good, ranging from 70-105%. Between- and within-assay coefficients of variation ranged from 6.3% to 13.8%, and 2% to 3.5%, respectively. Chlordiazepoxide chromatographed poorly (between assay coefficient of variation 35.4%, within-assay 7%), and we set the cut-off value for this compound at 5.0 mg/L.

Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

Urine benzodiazepines screening of involuntarily drugged and robbed or raped patients.

This study involved 35 patients who claimed to have been drugged before being robbed or raped, despite urine negative toxicologic screening by immunoenzymatic methods. The urines were frozen for further investigations, including enzymatic hydrolysis of urinary conjugates, liquid-solid extraction and, finally, immunoenzymatic screening of concentrated urine extract. Urine benzodiazepines were analyzed by immunoenzymatic assay before and after enzymatic hydrolysis combined with extraction. On direct immunoenzymatic screening, 17 of the 35 urine samples were benzodiazepine positive. Enrichment of preserved specimens improved the detection threshold from 200 ng/mL to 50 ng/mL and 10 of the 18 negative urines became positive. This method allowed us to demonstrate the benzodiazepines in half of previously negative urine samples. Benzodiazepine screening is particularly problematic because of low dosage, rapid elimination, failure to detect conjugated metabolites by immunoenzymatic reagents and high threshold of sensitivity for certain substances.

Effect of beta-glucuronidase on urinary benzodiazepine concentrations determined by fluorescence polarization immunoassay

In samples from patients treated with oxazepam, beta-glucuronidase increased the immunoreactivity of urinary benzodiazepines analyzed by fluorescence polarization immunoassay (FPIA). Increasing concentrations of beta-glucuronidase added to samples from drug-free controls did not influence the results. In the absence of beta-glucuronidase, 22 of 35 samples from patients undergoing detoxification gave positive results at a cutoff concentration of 200 micrograms/L. Pretreatment with beta-glucuronidase increased the number of drug-positive samples to 33. The drug-negative samples were obtained from two patients who had been oxazepam-free for at least 1 week. Thus, beta-glucuronidase can be used to increase the sensitivity of the urinary benzodiazepine FPIA without reducing the specificity of the method.

False-positive immunoassay results for urine benzodiazepine in patients receiving oxaprozin (Daypro)

False-positive immunoassay results for urine benzodiazepine in patients receiving oxaprozin (Daypro)

Determination of Triazolam and Its Metabolites in Human Urine by EMIT, Gas Chromatography-Mass Spectrometry and Thermospray Liquid Chromatography-Mass Spectrometry.

EMIT, gas chromatography-mass spectrometric (GC-MS) and thermospray high performance liquid chromatography-mass spectrometric (LC-MS) methods for the determination of triazolam (TRZ) and its metabolites (1-hydroxymethyltriazolam (α-HT) and 4-hydroxytriazolam (4-HT)) in the human urine were investigated. TRZ was analyzed by EMIT st Urine Benzodiazepine Assay. EMIT cutoff was 50 ng/ml of TRZ. TRZ and its metabolites were extracted from the human urine using Sep-Pak C18 cartridge with dichloromethane-methanol (90 : 10 v/v) as the eluent. The eluate was evaporated to dryness under stream of N2. The residue was dissolved in 1 ml of methanol, 50 μl was injected into the LC-MS. LC analyses were performed on a TSKgel Octyl-80Ts in the solvent system of 100 mM ammonium acetate-methanol (50 : 50 v/v) at a flow rate of 1.0 ml/min. For GC-MS, the extract was derivatized at 90°C for 30 min with 50 μl BTZ. GC analyses were performed on a fused silica column DB-1. The column temperature was 290°C. The detection limits of these compounds by scan mode were 800 ng/ml for α-HT and 4-HT with GC-MS, 50 ng/ml for TRZ and α-HT, 100 ng/ml for 4-HT with LC-MS and those by selected ion monitoring mode were 50 ng/ml for α-HT, 100 ng/ml for 4-HT with GC-MS, 5 ng/ml for TRZ and α-HT, 10 ng/ml for 4-HT with LC-MS.

Optimal enzymatic hydrolysis of urinary benzodiazepine conjugates.

Conditions for the enzymatic hydrolysis of benzodiazepine conjugates were systematically examined. Optimal recovery of the free drugs occurs when 1 mL of urine buffered to pH 4.5 is incubated with 5000 U of Helix pomatia beta-glucuronidase at 56 degrees C for 2 h. Urine specimens containing conjugates of temazepam, oxazepam, lorazepam, alpha-hydroxyalprazolam, 2-hydroxyethylflurazepam, and N-desalkyl-3-hydroxyflurazepam as targeted benzodiazepines were used. The freed drugs were quantitated by gas chromatography-mass spectrometry.

Analysis of urinary benzodiazepines using solid-phase extraction and gas chromatography-mass spectrometry.

A solid-phase extraction and GC-MS confirmation method was developed for certain urinary diazolo- and triazolobenzodiazepines, including the metabolites of lorazepam, clonazepam, alprazolam, and triazolam. The latter two do not form benzophenones, and the others are not readily confirmed by conventional thin-layer chromatography or GC-MS techniques. Samples were hydrolyzed with glucuronidase at 37 degrees C, adjusted to pH 4.5, extracted with Bond Elut Certify columns, dried, and derivatized using BSTFA with 1% TMCS. Sample preparation time averaged 4 hours. A GC-MS selected-ion-monitoring acquisition method targeting retention time, molecular ion abundances, and qualifier ion ratios was used to determine positive results. The recovery of 7-NH2-clonazepam was 95%, and recoveries of alpha-hydroxyalprazolam, alpha-hydroxytriazolam, and lorazepam were greater than 66%. Linearity was demonstrated from 0.1 to 1.0 microgram/mL for each drug. Within-run CVs were less than 11%, and between-run CVs were less than 16%. Using this technique, we have been able to confirm suspected cases of abuse that had not been confirmed by previous techniques.

Confirmation of benzodiazepines in urine as trimethylsilyl derivatives using gas chromatography—mass spectrometry

A confirmation procedure for the identification and quantitation of various benzodiazepines in urine is presented. The urine sample is first hydrolyzed enzymatically because of the glucuronide conjugation of some benzodiazepine metabolites, then extracted using bonded-phase columns. After elution into an organic solvent, the samples are evaporated, converted to the trimethylsilyl ether derivatives and analyzed by electron ionization GC—MS. Quantitation was performed using selected-ion monitoring for each benzodiazepine using prazepam as the internal standard. The method provides excellent linearity and sensitivity for the trimethylsilyl derivatives.

Benzodiazepine analysis by negative chemical ionization gas chromatography/mass spectrometry.

The application of gas chromatography/mass spectrometry (GC/MS) for the simultaneous quantitation of seven commonly encountered urinary benzodiazepine metabolites is described. After comparison of the signal-to-noise ratios of high mass ions of benzodiazepines using electron impact (EI), positive chemical ionization (PCI), and negative chemical ionization (NCI), NCI was chosen because of its increased sensitivity, which ranged from four to several thousand times that of either PCI or EI. This method is novel because NCI spectra for many of these compounds have not been described. For quantitation of benzodiazepines in urine, sample preparation consisted of enzymatic hydrolysis, liquid-liquid extraction, and reaction with a silylating reagent to form trimethylsilyl derivatives. The extraction efficiency of the method was greater than 70% (range, 73-89%) for nordiazepam, oxazepam, temazepam, lorazepam, N-1-hydroxyethylflurazepam, alpha-hydroxyal-prazolam, and alpha-hydroxytriazolam; the linear range for these compounds was from 50 to 2000 ng/mL. Within-run precision was less than 6% for all analytes in the range 50-2000 ng/mL; however, run-to-run precision ranged from 3 to 21%, depending on the analyte and concentration. Quantitation was based on area ratio of high mass ions relative to deuterated internal standards, acquired by scanning the mass range from m/z 250 to 450. Because these studies were performed in the scan mode, if desired, the sensitivity could be increased by using selected ion monitoring.

Urinary screening for alpha-OH triazolam by FPIA and EIA with confirmation by GC/MS.

Triazolam is a very short-acting triazolobenzodiazepine with sedative-hypnotic properties. Approximately 2% of an oral dose is excreted unchanged in the urine. The major urinary metabolite is alpha-hydroxytriazolam glucuronide (70% of the dose). The objective of this study was to characterize the reactivity of alpha-hydroxytriazolam in the urine benzodiazepine assay by fluorescence polarization immunoassay (FPIA; Abbott TDx) in comparison with enzyme immunoassay (EIA; Syva EMIT d.a.u. benzodiazepine assay). alpha-OH triazolam at 300 ng/mL gave a response equivalent to the 200-ng/mL nordiazepam Abbott calibrator. In the EMIT assay, alpha-OH triazolam gave a response equivalent to the 300-ng/mL calibrator (Syva) at 100-200 ng/mL. Both immunoassays gave positive results in 9 out of 9 urine specimens collected from individuals receiving triazolam. Confirmation was performed by analyzing for alpha-OH triazolam after enzymatic hydrolysis and formation of a TMS derivative for GC/MS. All urine specimens were positive for alpha-OH triazolam. In conclusion, both the FPIA and EIA immunoassay screening assays are acceptable for detecting the presence of alpha-OH triazolam in the urine of patients receiving therapeutic doses of triazolam.

Confirmation of low concentrations of urinary benzodiazepines, including alprazolam and triazolam, by GC/MS: an extractive alkylation procedure.

Urine samples containing diazolo- and triazolobenzodiazepines and metabolites were hydrolyzed with beta-glucuronidase and extracted with methylene chloride. The extracts were treated with methyl iodide, methylene chloride, and tetrahexylammonium hydrogen sulfate in basic solution to form the methyl derivatives of the drugs and metabolites. GC/MS analysis resulted in the following test characteristics: day-to-day precision at 360 ng/mL (120 ng/mL for the triazolobenzodiazepine metabolites) was 2.4 to 5.5% CV; calibration curves were linear to 6000 ng/mL (1000 ng/mL for the triazolobenzodiazepine metabolites), and operational limits of quantitation were in the range 13-25 ng/mL.

Immunologische Untersuchungen auf Benzodiazepine—Der Einfluß von Strukturmerkmalen auf die Kreuzreaktivität ‐

The manufacturers' descriptions for immunoassays for the detection of benzodiazepines in urine suggest that all therapeutically used benzodiazepines are detected equally. It is unlikely that the different molecular structures do not influence the results of pertinent measurements. Therefore, the cross-reactivity of 35 benzodiazepines and benzodiazepine-metabolites was determined with the TDx-system. As compared to nordiazepam all compounds--except diazepam--had a reduced cross-reactivity. In order to get a TDx result equal to or exceeding 200 ng/ml nordiazepam-equivalents, high benzodiazepine concentrations in urine are required in some cases. These high concentrations are not obtained in the therapeutic range of application. These considerations can be applied to other immunoassays like RIA and EMIT.