7-amino Metabolites Research Articles

B-271 High Sensitivity Homogeneous Enzyme Immunoassay for Benzodiazepines and Metabolites

Abstract Background Benzodiazepines are widely prescribed for treating various conditions such as anxiety, insomnia, and seizures due to their sedative and anxiolytic properties. Despite their therapeutic benefits, concerns persist regarding their potential for misuse, dependence, and addiction. Urine drug screening immunoassays serve as an important tool in monitoring benzodiazepine usage, ensuring adherence to prescribed regimens, identifying misuse or abuse, and facilitating necessary medical interventions. However, limitations of benzodiazepine immunoassays are well-documented in literature, with issues surrounding their lack of sensitivity to glucuronidated and 7-amino metabolites, inability to detect newer benzodiazepines, and poor cross-reactivity with different benzodiazepines. ARK Diagnostics has developed a highly sensitive homogeneous enzyme immunoassay capable of detecting both classic and designer benzodiazepines, in addition to their glucuronide and 7-amino metabolites in human urine at a cutoff concentration 200 ng/mL, without the need for glucuronidase or sample pretreatment. Methods The ARKTM Benzodiazepine Plus Assay is a liquid-stable homogenous enzyme immunoassay consisting of two reagents. The assay uses temazepam as the 200 ng/mL cutoff calibrator. The performance characteristics of this assay, including precision, spiked recovery, specificity, and method comparison to LC-MS/MS, were evaluated on the Beckman Coulter AU680 automated clinical analyzer. Results The ARKTM Benzodiazepine Plus demonstrated acceptable precision, with no overlap between cutoff (200 ng/mL) and ±25% control levels (150 ng/mL and 250 ng/mL) in a histogram overlap analysis, and ≤3.6% CV in semi-quantitative mode. Spiked temazepam samples spanning the semi-quantitative assay range of 1000 ng/mL were recovered between 95.0% and 103.7% of the spiked levels. Thirty-eight benzodiazepines produced cross-reactivities >80%, including the following benzodiazepines and metabolites: lorazepam, clonazepam, diazepam, alprazolam, temazepam, oxazepam glucuronide, lorazepam glucuronide, temazepam glucuronide, 7-aminoclonazepam, and 7-aminoflunitrazepam. In method comparison studies, a total of 103 urine samples containing benzodiazepines with LC-MS/MS values ranging from 1.3 ng/mL to 3257.6 ng/mL were tested with the ARKTM Benzodiazepine Plus Assay. Eighty samples tested positive with values ≥200 ng/mL and 23 samples tested negative with values <200 ng/mL by the ARKTM Benzodiazepine Plus assay. Of the 23 samples that tested negative, 21 had LC-MS/MS values <200 ng/mL and 2 samples were identified as midazolam samples with metabolite a-OH-midazolam levels >200 ng/mL. Conclusions The ARKTM Benzodiazepine Plus Assay enables the sensitive, rapid, and reliable measurement of benzodiazepines and their metabolites in human urine, without the need for hydrolysis or pretreatment. The assay addresses the present constraints of currently available benzodiazepine immunoassays, including their insufficient sensitivity, limited cross-reactivity to metabolites, and the requirement for glucuronidase pretreatment. The ARKTM Benzodiazepine Plus Assay is applicable to a wide range of clinical chemistry analyzers.

Intralipid as a matrix additive for evaluating hyperlipidemic postmortem blood.

Postmortem whole blood samples can differ greatly in quality where hyperlipemia is a frequent variable that can influence the results of analytical methods. The aim of this study was to investigate the influence of lipemia on postmortem analysis as well as demonstrate the usage of Intralipid in comparison to pooled postmortem lipids as matrix additives for meaningful evaluation and validation of hyperlipidemic postmortem samples. Hyperlipidemic blood samples were simulated by adding different concentrations of Intralipid or pooled authentic postmortem lipids to bovine whole blood. The hyperlipidemic blood samples were spiked with 14 benzodiazepines and five sedative and antianxiety drugs (alprazolam, clonazepam, 7-aminoclonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, hydroxyzine, lorazepam, midazolam, nitrazepam, 7-aminonitrazepam, nordazepam, oxazepam, propiomazine, dihydropropiomazine, temazepam, triazolam, zolpidem and zopiclone). Samples were prepared with liquid-liquid extraction followed by ultra-high performance liquid chromatography-mass spectrometry. The effects of lipemia on the recovery of analytes and internal standards (ISs) were evaluated to determine the effect of, and any differences between, the two additives. Lipemia was found to cause major interference when quantifying the analytes. For most analytes, the ISs could compensate for analyte losses. However, the most hydrophilic analytes (7-amino metabolites), together with the most lipophilic analytes (propiomazine and dihydropropiomazine), were greatly affected by lipemia (<50% recovery), and the IS could not compensate for analyte losses. In general, lower analyte recoveries were observed for samples with Intralipid as a lipemic additive in comparison to those containing pooled postmortem lipids. Both Intralipid and pooled postmortem lipids showed marked effects on the analytical results. Intralipid gave a good indication of the effects of lipemia and could be a useful tool for making a meaningful evaluation of hyperlipidemic postmortem samples during the method development and validation.

Comparative Study of Postmortem Concentrations of Benzodiazepines and Z-Hypnotics in Several Different Matrices.

Benzodiazepines and z-hypnotics are detected in the majority of fatal overdose cases in Norway, often in combination with other drugs of abuse, and their concentrations in peripheral blood (PB) might be important to elucidate the cause of death. In some forensic autopsies, PB is however not available. The aim of the present study was to compare concentrations of benzodiazepines and z-hypnotics in five alternative matrices to assess whether these concentrations are comparable to concentrations in PB. A total of 109 forensic autopsy cases were included. PB, cardiac blood (CB), pericardial fluid (PF), psoas muscle (PM), lateral vastus muscle (LVM) and vitreous humor (VH) from each case were analyzed using ultra high performance liquid chromatography--tandem mass spectrometry. We were able to detect clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, nitrazepam, 7-aminonitrazepam, diazepam, nordiazepam, oxazepam, alprazolam, midazolam, zopiclone and zolpidem in all the analyzed matrices. Concentrations measured in VH were generally much lower than those of PB for all compounds except zopiclone. 7-Amino metabolite concentrations were high compared to the parent compounds, although less so for the muscle samples. Concentrations of the parent nitrobenzodiazepines in muscles were higher than those in PB, but for the other compounds, concentrations in muscle showed good correspondence with PB. Both CB and PF were viable alternative matrices for PB, although a larger variation and a tendency for higher concentrations in PF were observed. This study shows that CB, PM, LVM and PF can give comparable concentrations to PB for benzodiazepines and z-hypnotics, while VH was less suitable. The concentrations in alternative matrices must, however, be interpreted carefully.

Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC-MS/MS.

Collection of oral fluid (OF) is easy and non-invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high-throughput ultra-high-performance liquid chromatography-tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96-well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry-over. Inter-assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7-amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium-labelled and 13 C-labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12-month period. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of Nitrobenzodiazepines and Their 7-Amino Metabolites in Oral Fluid.

Clonazepam, nitrazepam and flunitrazepam are frequently used benzodiazepines, both as prescribed medication and as drugs of abuse. Little is, however, known about how these drugs are excreted in oral fluid. It has been claimed that the parent drugs are more likely to be detected in oral fluid than the 7-amino metabolites. The aim of this study was to investigate whether the parent drugs or the 7-amino metabolites of the nitrobenzodiazepines were most frequently detected in authentic oral fluid samples. Oral fluid samples were collected from patients undergoing opioid maintenance treatment. Cases where clonazepam, nitrazepam, flunitrazepam and/or their metabolites were detected were included. The samples were collected using the Intercept Oral Specimen Collection Device. A cutoff concentration of 1 nM (∼0.3 ng/mL) in oral fluid-buffer mixture was applied for all the substances. A total of 1,001 oral fluid samples were positive for clonazepam and/or 7-aminoclonazepam; both substances were detected in 707 samples, only the parent drug in 64 cases and only the metabolite in 230 cases. For nitrazepam, both substances were detected in 139 samples; only the parent drug in 16 cases and only the metabolite in 56 cases. Flunitrazepam only was not detected in any sample; both substances were detected in one of these cases, and only the metabolite in three cases. This study revealed that 7-amino metabolites were more likely to be detected in oral fluid than the parent drugs.

Postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in body fluids and solid tissues in an autopsy case: Usefulness of bile for their detection

We experienced an autopsy case of a woman in her 70s, in which the direct cause of her death was judged as asphyxia due to the occlusion of food in the trachea. The postmortem interval was estimated at about 2days. The specimens dealt with were femoral vein blood, right heart blood, left heart blood, bile, brain, lung, heart muscle, liver, spleen, kidney, pancreas, skeletal muscle, and adipose tissue. By tentative drug screening, we found a high concentration of 7-aminoflunitrazepam in the femoral vein blood, which lead us to examine the postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in her body fluids and solid tissues. The extraction of flunitrazepam, 7-aminoflunitrazepam and internal standard nimetazepam was performed by a modified QuEChERS method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, we used the standard additional method to overcome the matrix effects. The concentration of 7-aminoflunitrazepam were generally much higher than those of the parent drug flunitrazepam for most specimens except for the adipose tissue, showing that flunitrazepam is readily metabolized to its 7-amino metabolite after absorption into the body both antemortem and postmortem. The outstandingly highest concentration of 7-animoflunitrazepam was found in the bile, followed by the kidney, pancreas, left heart blood, spleen and liver. Although a majority of flunitrazepam was converted to 7-aminoflunitrazepam, the flunitrazepam concentration was highest in the pancreas, followed by the spleen, bile, left heart blood, and brain. In contrast to the results on synthetic cannabinoids, the levels of flunitrazepam and 7-animoflunitrazepam in the adipose tissue were relatively low. The present study showed that the bile may be a useful specimen for detection of unchanged benzodiazepines/their metabolites to be collected at autopsy.

Hair analysis by liquid chromatography–tandem mass spectrometry in toxicological investigation of drug-facilitated crimes: Report of 128 cases over the period June 2003–May 2004 in metropolitan Paris

In recent years, reports of drug-facilitated crimes (DFC) have been increasing. The drugs involved are sometimes difficult to detect, because of their low dosages and the long time ellapsed between alleged DFC and blood and urine sampling. In order to detect benzodiazepines and benzodiazepine-like hypnotics, we developed an approach for hair analysis by liquid chromatography–tandem mass spectrometry using a triple stage quadrupole with an electrospray ionization (LC–ESI-MS/MS). Separation was performed on an Uptisphere ODB C18 column using a gradient of 2 mM formate buffer and acetonitrile. For the 23 compounds studied, detection limits are lower than 2 pg/mg, but a specific extraction procedure is needed for 7-amino metabolites. Over a 1-year period within the city limits of Paris and three suburbs, we tested blood and urine from victims of sexual assaults, robbery and battery in which psychoactive substances were suspected of being involved. Hair was collected 4–8 weeks after the alleged DFC. Over the 128 cases studied, results of simultaneous analysis of blood, urine, and hair allowed us to conclude that 23 cases were real DFC. In 18 cases, no conclusion was possible since no hair was sampled and/or results were negative. In 56 cases, victims were previously using narcotics, cannabis, and/or a prescribed drug, according to the compounds detected in hair strands. Thirty-one cases were not DFC cases. This study indicates that the prevalence of zolpidem and clonazepam is high, followed by bromazepam, nordazepam, and midazolam. Others benzodiazepines and analogs are rare. LC–ESI-MS/MS is a good tool for toxicological investigations of DFC. Testing blood, urine, and hair by this technique may reveal drug presence, even if it was administered at a single therapeutic dose. That may be helpful to prosecute perpretators or to exclude a drug-facilitated crime.

Stability of Nitrobenzodiazepines in Postmortem Blood

Studies were undertaken to determine the stability of nitrobenzodiazepines and their 7-amino metabolites in water and blood. At 22 degrees C nitrazepam and clonazepam were stable in sterile fresh blood containing preservative over 28 days, whereas 25% of flunitrazepam was degraded. At 37 degrees C all three drugs were substantially lost over 9 h (29-51%). There was only a small loss observed for the 7-amino metabolites and no substantial amounts of parent drug and 7-amino metabolite were degraded in water under these conditions. In the absence of preservative substantial amounts (25-50%) of parent drugs were lost in fresh blood over 10 days at 22 degrees C. In bacterially-contaminated postmortem blood all three drugs were completely degraded over 8 h at 22 degrees C with almost all drug completely converted to the respective 7-amino metabolite. These metabolites were also partially degraded (10-20%) over 45 h at 22 degrees C. All 3 nitrobenzodiazepines were stable in blood stored for up to 24 months at -20 degrees C, or 4 degrees C over 10 months. Their respective 7-amino metabolites were, however, relatively unstable at -20 degrees C with a significant loss (29%) after 2 months. At 4 degrees C a 21% loss occurred after 1 month. Freeze/thawing was found not to affect the concentration of nitrobenzodiazepine and 7-amino metabolites. These results show that the nitrobenzodiazepines and their metabolites are unstable chemically and metabolically in blood. We advise that blood collected for the purpose of nitrobenzodiazepine determinations should be preserved with sodium fluoride, stored at -20 degrees C and assayed as soon as practicable, preferably within a week of collection.

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.

High-performance liquid chromatographic procedure for the measurement of nitrobenzodiazepines and their 7-amino metabolites in blood.

A simple and sensitive HPLC method is described for the determination of the nitrobenzodiazepines, nitrazepam, flunitrazepam and clonazepam and their respective 7-amino metabolites in post-mortem blood. Using a single-step extraction the nitrobenzodiazepines were recovered from 0.5 ml of blood using butyl chloride. The solvent was evaporated to dryness and the reconstituted residue injected onto an HPLC system. Separation was achieved using a phenyl-bonded reversed-phase column with an acetonitrile-phosphate buffer mobile phase. Ultraviolet detection (240 nm) was used for quantitation. A linear response was observed between 0.01 and 0.60 mg/l. Assay precision between and within assays was less than 20% for all analytes. The limits of detection ranged from 0.001 to 0.008 mg/l with a limit of quantitation of 0.01 mg/l for all analytes.

Quantitation of clonazepam and its 7-amino and 7-acetamido metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method is reported for the determination of clonazepam and its metabolites 7-amino- and 7-acetamidoclonazepam. Extraction from buffered plasma is carried out at pH 9.5 with hexane—ethyl acetate (7:3) for clonazepam and with chloroform for the metabolites. Flunitrazepam, 7-aminodemethylflunitrazepam and 7-acetamidoflunitrazepam are used as the internal standards for clonazepam and its 7-amino and 7-acetamido metabolites, respectively. To prevent decomposition of 7-aminoclonazepam a high concentration of 7-aminomethylclonazepam is added to the plasma. Chromatography is carried out on a reversed-phase column with detection at 254 nm for clonazepam and 240 nm for the metabolites. Using the method it was possible to determine 5 ng/ml clonazepam, 7-aminoclonazepam and 7-acetamidoclonazepam in plasma with coefficients of variation of 9.5%, 5.9% and 8.9%, respectively. This method can be used to measure clonazepam in plasma from patients treated with other antiepileptics. It may also be utilized for in vitro studies on the metabolism of clonazepam in subcellular fractions from the liver.

Kinetics of biotransformation of clonazepam to its 7-amino metabolite in the monkey.

The pharmacokinetic behavior of the 7-amino metabolite of clonazepam administered exogenously and formed endogenously from the parent drug was studied in a group of rhesus monkeys using constant rate intravenous infusions. Plasma levels of the 7-amino metabolite and/or clonazepam were determined with a GC-CI-MS method. The biological half-life of the 7-amino metabolite (2.2 +/- 1.0 hr) was shorter than that of clonazepam (4.9 +/- 0.2 hr). Total body clearance of the metabolite (0.83 +/- 0.16 liters/hr/kg) was larger than that of the parent drug (0.55 +/- 0.09 liters/hr/kg). The kinetics of in vivo biotransformation were described by a two-compartment model in which formation and disposition of the metabolite follow first-order processes. The fraction of a dose of clonazepam appearing in the systemic circulation as 7-amino metabolite was 0.70 +/- 0.30. This value may underestimate the actual fraction formed, if the metabolite is susceptible to first-pass metabolism following in situ formation.

Determination of clonazepam and its amino and acetamido metabolites in human plasma by combined gas chromatography chemical ionization mass spectrometry and selected ion monitoring

Our previously published gas chromatography chemical ionization mass spectrometry stable isotope dilution assay for clonazepam and its 7-amino metabolite in plasma has been modified to obtain more consistent recoveries of the amine metabolite and to permit the determination of the 7-acetamido metabolite of clonazepam. In addition, the manuscript describes the details of a method for a least squares analysis of the nonlinear standard curves associated with the use of certain stable isotope labeled analogs as internal standard.

Determination of clonazepam and its 7-amino metabolite in plasma and blood by gas chromatography—chemical ionization mass spectrometry

A sensitive gas chromatography, ammonia chemical ionization mass spectrometry, 15N isotope dilution assay has been developed to measure clonazepam and its 7-amino metabolite in blood or plasma. The method was used to measure both compounds in the blood of one subject administered a single 2-mg dose of clonazepam, and in the plasma of thirteen subjects on a clonazepam oral dosing regimen.

Radioimmunoassay of the Anticonvulsant Agent Clonazepam

A simple and specific radioimmunoassay was developed for the determination of the anticonvulsant agent clonazepam directly in plasma without extraction. Antibodies to clonazepam were produced in rabbits after immunization with an immunogen prepared by covalently linking the 3-hemisuccinyloxy derivative of clonazepam to bovine serum albumin. When employing 3H-clonazepam as the tracer, the radioimmunoassay has a limit of sensitivity of 5 ng/ml using a 0.1-ml sample of plasma. The antibodies exhibited a high degree of specificity for clonazepam; no cross-reactivity was observed with its 7-amino and 7-acetylamino metabolites nor with a number of other widely prescribed anticonvulsant agents that might be administered in conjuction with clonazepam. Satisfactory agreement was obtained for the plasma levels of clonazepam in humans when samples were assayed by the radioimmunoassay and an established electron-capture GC technique. By virtue ot its simplicity, the radioimmunoassay offers a distinct advantage to the clinician for monitoring plasma clonazepam levels and the compliance of patients undergoing anticonvulsant therapy with the drug.