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A Study on Chromatography Methods for the Separation and Detection of Certain Benzodiazepine Drug in Forensic Sample
Abstract
Forensic Scientists are required to identify an ever increasing and more complex assortment of drugs and related compounds. A rapid, sensitive and specific thin layer chromatography (TLC), high-Performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) methods were utilized for the analysis of certain benzodiazepines (BZDs). These drugs were once an occasional problem, today they have become much more common. Due to the structural similarity of the specimens encountered by the forensic laboratory, an array of instruments is needed to correctly identify these substances. Any of the BZDs can be identified by combining the results obtained with different mobile phases. HPLC proposes a cost efficient method with the ruggedness and consistency necessary for forensic testing and consequently is widely used in forensic laboratories today. GC-MS is one of the most commonly used techniques for the identification and quantitation of forensic drug samples. As a “hyphenated” technique, it combines the separation power of a GC with the analyte specificity of a spectroscopic technique, provided that vastly specific spectral data on individual compounds in a complex mixture often devoid of prior separation. We succeeded in the separation of the encountered BZD drug diazepam in the forensic sample. The method was validated for linearity, accuracy, precision and limit of detection. A competent forensic toxicologist relies on their own case experience as well as the unique state of affairs of each case under assessment.
Keywords: Benzodiazepines; Diazepam; Separation; Detection; TLC; HPLC; GC-MS
... Clonazepam is a benzodiazepine derivative that is widely used as a treatment for sleep disorders, anxiety, muscle relaxant, sedative, and epileptic seizures. It has side effects such as drowsiness and fatigue when low doses lead to dizziness, mood swings, and memory loss at high concentrations (Gurusamy and Thangadurai, 2019;Degreef et al. , 2021;Qriouet et al., 2019) . ...
An easy and sensitive spectrophotometric method has been suggested for the assay of clonazepam (CLZM) as pure form and in its formulation as tablets. The present method based on the reaction of CLZM with alizarin red S reagent (ALRS) via proton transfer reaction to form a colored solution with a maximum absorption at wavelength of 528 nm and the optimal conditions for the reaction were studied. The linearity was within the concentration range from 1 to 30 µg/ml, with an R 2 value (determination coefficient) of 0.9888 and the sensitivity express via the molar absorption of 7.04x10 3 l mol-1 .cm-1 , while Sandell's sensitivity index has been determined and equal to 0.0448 µg/ cm 2 .The percentage of relative standard deviation (RSD%) which express the precision of the method, percentage of relative error (RE%) express the accuracy, the LOD and LOQ also have been estimated. The application of the suggested method to the determination of CLZM in tablet gave satisfactory results.
Benzodiazepines are an important class of drugs commonly administered with a potential for abuse and environmental pollution. This review focuses on the liquid chromatographic electrochemical detection of the benzodiazepine class of drugs. These are characterised by a readily electrochemically reducible azomethine group, with a number also substituted by other electrochemically active groups. Liquid chromatography employing both single and dual electrode detection has been reported for a variety of benzodiazepines and their metabolites in biological, pharmaceutical, biomedical and forensic investigations. Recently, electrochemistry has been utilised to mimic biological oxidation processes and has been combined with liquid chromatography/mass spectroscopy fr their identification and quantification of the products generated. The present review fcuses on recent developments in liquid chromatographic- electrochemical determination of benzodiazepines reported since 2006, with earlier reports given in summary.
Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability.
A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, α-hydroxyalprazolam,
α-hydroxyethylflurazepam, α-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam,
oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium.
Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system.
The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization
mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover.
Average recovery and imprecision (within-run, between-run, and total % CV) were within ± 15% of the target concentrations
for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within ± 20% for meconium (10 to 5000 ng/g). In all,
205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS
results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium
specimens.
A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.
We report a midazolam-related death that occurred during endoscopic retrograde cholangiopancreatography (ERCP). The acute intoxication due to midazolam overdose was confirmed by high-pressure liquid chromatography (HPLC) analysis of the blood samples taken from the patient in the intensive care unit (2.8 microg/ml) and postmortem (2.4 microg/ml). The case strongly emphasizes the necessity of the precautions that should be taken when midazolam is intravenously administered.
There is a continued high prevalence of benzodiazepine use by older community-residing adults and of their continued prescription by practitioners, despite well known adverse effects and the availability of safer, effective alternatives.
To understand factors influencing chronic use of benzodiazepines in older adults.
Qualitative study, semistructured interviews with physicians.
Thirty-three practicing primary care physicians around Philadelphia.
Qualitative interviews were audiotaped, transcribed, and entered into a qualitative software program. A multidisciplinary team coded transcripts and developed themes.
Physicians generally endorsed benzodiazepines as effective treatment for anxiety, citing quick action and strong patient satisfaction. The use of benzodiazepines in older adults was not seen to be problematic because they did not show drug-seeking or escalating dose behavior suggesting addiction. Physicians minimized other risks of benzodiazepines and did not view monitoring or restricting renewal of prescriptions as an important clinical focus relative to higher-priority medical issues. Many physicians expressed skepticism about risks of continued use and considerable pessimism in the successful taper/discontinuation in older patients with long-term use and prior failed attempts. Physicians also anticipated patient resistance to any such efforts, including switching physicians.
Primary care physicians are averse to addressing the public health problem of benzodiazepine overuse in the elderly. Their attitudes generally conflict with practice guidelines and they complain of a lack of training in constructive strategies to address this problem. A 2-pronged effort should focus on increasing skill level and preventing new cases of benzodiazepine dependency through improved patient education and vigilant monitoring of prescription renewal.
A simple and rapid procedure for the simultaneous screening of 14 benzodiazepines in oral fluid is presented. The procedure
involves a solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The target compounds
include diazepam, oxazepam, temazepam, nordiazepam, lorazepam, chlordiazepoxide, alprazolam, α-hydroxyalprazolam, desalkylflurazepam,
hydroxyethylflurazepam, clonazepam, 7-aminoclonazepam, flunitrazepam, and 7-aminoflunitrazepam. Oral fluid was obtained using
a simple device that collects approximately 0.4 mL of oral fluid and dilutes it with 0.8 mL of preservative. The oral fluid
sample preparation involves a solid-phase extraction on a Varian Bond Elut cartridge. Quantitation was performed by LC-MS-MS
using nordiazepam-d5 as the internal standard. The extraction efficiency exceeded 83% for all compounds except for 7-aminoclonazepam, which had
a recovery of 55%. The limits of quantitation ranged from 0.1 ng/mL to 1.0 ng/mL in the multiple reaction monitoring mode.
This method was used to confirm 41 patients that screened positive using the OraSure Technologies Benzodiazepine Intercept®
MICRO-PLATE Enzyme Immunoassay kit. All screened-positive patients were positive for at least one of the analyzed benzodiazepines,
thus showing that this method is suitable for confirmation of the Intercept Benzodiazepine assay.
We report a new, sensitive, fast, precise, reversed phase-high performance liquid chromatography method for the determination of flurazepam in (capsule) dosage form. The developed method is validated by measuring the linearity, precision, limit of detection (LOD), robustness and ruggedness, drug recovery and the system suitability parameters. The HPLC conditions are; methanol:acetonitrile (80:20 v/v) mobile phase, Chromosil C 18 column (250 mm × 4.6 mm × 5 μm) stationary phase, pump pressure (6.5 MPa), flow rate (0.9 ml/min) and the detection wavelength of 230 nm. The measured flurazepam retention time is 5.27 minutes. The limit of detection is 0.05 μg/ml. The linearity range measured is from 20-200 μg/mL with a correlation coefficient (R ²=0.9991). The measured parameters indicate the developed method is useful in determination of flurazepam in pure and capsule dosage form.
A simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of clobazamin tablet dosage form. Isocratic elution at a flow rate of 1ml min -1 was employed on a symmetry C18 column at ambient temperature. The mobile phase consisted of Methanol:water:OPA(0.1%) 35:40:25 (v/v/v). The UV detection wavelength was at 239nm.Linearity was observed in concentration range of 4-16ppm. The retention time for Clobazam was 4.71 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Clobazam in pharmaceutical dosage forms.
Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negative-ion chemical ionization (NICI) mass spectra of 24 benzodiazepines are presented. In the PIEI mode, peaks due to M, M − H, M − CO and a liberated R3-phenyl ring appeared in many compounds. The peaks at M − 47 and 56 or 70 were characteristic for nitro- and oxazolo-compounds, respectively. In the PICI mode, most spectra showed the base peaks due to M + H and small peaks due to M + C2H5; small peaks due to M − R1 or M − R1 + 2H also appeared for many compounds. For the nitro-compounds, peaks at M − 29 commonly appeared. For the oxazolo-compounds, peaks due to the loss of the R3-phenyl ring appeared. In the NICI mode, either molecular or [M − H]− quasi-molecular anions were the base peaks in 10 compounds. Small anions at M + 15 (M − H + O) also appeared in many compounds. Anions due to liberated halogens were observed for most compounds; bromine gave its base peaks. An procedure for benzodiazepines from human urine and plasma, and their separation by gas chromatography (GC) with a wide-bore capillary column are also presented to serve for their actual identification by GC/mass spectrometry (MS). The wide-bore capillary column was useful to prevent the drugs from decomposition.
An accurate and reproducible method for the analysis of flurazepam hydrochloride in pharmaceutical preparations has been described. A simple and rapid isocratic HPLC elution method was employed which requires about 15 minutes to be performed. The percentage mean recovery of flurazepam hydrochloride in Dalmane capsules was found to be 100.67% + 1.23% of the declared amount
A blood plasma sample was spiked with flurazepam and its metabolites and diluted with water (1 + 1); after protein precipitation, flurazepam and three of its metabolites were extracted into ethyl acetate at pH 9. After evaporation of the solvent, the residue was dissolved in the mobile phase for injection onto a reverse-phase LiChrosorb column; eluents were methanol-water (62:38) for separation of the metabolites and methanol-phosphate buffer (85:15) for flurazepam. The limit of detection varied from 0.1 to 1.6 ng. Recoveries from spiked plasma (1 μg ml−1) were 94–95%. At 254 nm, the response was usually linear over the range 5–50 ng.
Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid-liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.
In recent years the need for rapid, sensitive and specific assays for benzodiazepines has resulted in the publication of a number of high-pressure liquid chromatographic (HPLC) methods for their determination. This paper reviews the methods available to date for the determination of chlordiazepoxide, clonazepam, diazepam, flurazepam, lorazepam, nitrazepam, oxazepam and their metabolites in biological fluids.
A reversed-phase high-performance liquid chromatographic method is described for the quantitative determination of alprazolam in the plasma of geriatric patients in the presence of 4-hydroxyalprazolam, alpha-hydroxyalprazolam, bromazepam, oxazepam, lorazepam, clobazam, desmethylclobazam, diazepam and desmethyldiazepam. The procedure is based on the enrichment of alprazolam on a PRP-1 pre-column, followed by the transfer of the compound in a forflush mode to the analytical column. Alprazolam can be quantified reliably down to a minimum concentration of 1 ng/ml of plasma.
A high-performance liquid chromatographic method was developed for the determination of alprazolam (ALP) and its active metabolites, alpha-hydroxyalprazolam (AOH) and 4-hydroxyalprazolam (4OH) in human serum. During assay development, the instability of 4OH was revealed. Factors affecting stability of 4OH were then investigated. In this report, the assay methodology for the determination of ALP and AOH, the instability of 4OH, subsequent interference of 4OH breakdown products with AOH quantification, and factors affecting 4OH stability are described. The clinical significance of our findings are reported.
An overview of methods for the determination of benzodiazepines in biological media, based on the application of chromatographic techniques, is presented. A general discussion of the techniques in terms of stability, selectivity, validation, standardization, detection and sensitivity is given. No single technique can be claimed as the method of choice for benzodiazepines. Gas chromatography with electron-capture detection has some strong claims and shows generally good sensitivity and reproducibility. High-performance liquid chromatographic equipment is readily available in most laboratories. The ultimate choice of an assay method for benzodiazepines will be determined by the clinical application (routine monitoring, pharmacokinetics, overdose, forensic medicine) and by the characteristics of the benzodiazepine, the expertise of the analyst, the equipment available, the desired sensitivity and specificity and the time involved in method development or adaptation and validation.
We compared three chromatographic methods for the therapeutic control of clonazepam. The first method analyzes the underivatized benzodiazepine, direct gas chromatography (GC) method, using nitrazepam as internal standard and GC with an electron-capture detector (63Ni). This method was compared with a second one, in which the same compounds were converted to the respective methylated derivatives (GC of derivatized samples) and also quantified by GC. The two methods were then compared with a third procedure for clonazepam quantification, using high-performance liquid chromatography (HPLC). Statistical analysis of clonazepam concentrations detected in the plasma of 37 patients, treated with Rivotril, indicated that HPLC is the most precise method, showed just significant correlations among the three methods, and suggested that plasma concentrations of clonazepam, estimated by the direct GC method, are significantly lower than those determined by the other two methods. The direct GC method and HPLC have been proposed as reference methods for the therapeutic control of clonazepam, with options for use left to the resources available in various laboratories.
A sensitive isocratic high-performance liquid chromatographic method is described, which allows the precise and accurate quantification of flurazepam and four metabolites with a single determination. A pharmacokinetic study was performed on nine volunteers and the main pharmacokinetic data are reported. The method was used to demonstrate that monodesethylflurazepam and didesethylflurazepam are major metabolites in men. One more unidentified flurazepam metabolite was detected.
A method for simultaneous determination of clobazam (CBZ) and its active metabolite N-desmethylclobazam (DCBZ) in various biological samples by RP-HPLC with UV detection is described. The determination of both CBZ and DCBZ is performed without derivatization. The internal standard is diazepam. The method is rapid and simple with sensitivity limits of 10 ng/mL for both CBZ and DCBZ and is suitable for routine analysis as well as for animal studies.
Two patients who attempted suicide with alprazolam had markedly elevated serum concentrations but manifested only mild toxicity. Overdose with alprazolam appears much less likely to be life-threatening than overdose with the tricyclic antidepressants.
The use of high-performance liquid chromatography for therapeutic drug monitoring of clonazepam has previously been limited by low sensitivity and labor-intensive liquid-liquid extractions. The present method was developed employing a rapid solid-phase extraction, thus minimising sample workup and providing analytical sensitivity down to 2 micrograms/L using 1 ml of plasma. Plasma samples were loaded onto C18 solid-phase extraction columns, and clonazepam and its internal standard (methyl-clonazepam) were eluted with methanol, dried, and reconstituted in 130 microliters of mobile phase. Chromatographic separation was achieved using a 3-microns RP18 column at 40 degrees C and a mobile phase of 32% acetonitrile and 0.5% glacial acetic acid in distilled water at 0.5 ml/min. Detection was carried out using ultraviolet absorbance at 306 nm. Retention times for clonazepam and methyl-clonazepam were approximately 7 and 12 min respectively. Standard curves were linear over a range of 5-200 micrograms/L with intraassay coefficients of variation of 1.2 and 4.8% at 200 and 5 micrograms/L, respectively. Plasma concentrations measured in patient samples were not statistically different from those obtained using an established gas chromatographic method, and quality control specimens from the Heathcontrol EQA Scheme were consistently within +/- 1.2 SD of the group means. There was no chromatographic interference from other benzodiazepines or other drugs used for the treatment of epilepsy.
A study of 16 deaths associated with toxic concentrations of benzodiazepines during the period of 5 years leading up to July 1994 is presented. Cases where other drugs, including ethanol, had contributed to the death were excluded. All cases were subject to a full macroscopic and microscopic examination by pathologists, and all cases were subject to a full toxicological work-up. Preexisting natural disease was a feature of 11 cases. In the remaining five cases, death was caused solely by benzodiazepines. There were 14 suicides. Nitrazepam and temazepam were the most prevalent drugs detected, followed by oxazepam and flunitrazepam. Minimum toxic femoral blood concentrations of 7-aminonitrazepam, 7-aminoflunitrazepam, and oxazepam were estimated as 0.5, 0.2, and 2 mg/L, respectively. Relating these deaths to prescription rates in Victoria suggest that flunitrazepam may be inherently more toxic if misused than other benzodiazepines currently available on the Australian market.
Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.
Study has been undertaken to determine the stability of four benzodiazepines: clonazepam, midazolam, flunitrazepam and oxazepam in whole blood samples. Spiked blood was stored at four different temperatures (room temperature, 4 degrees C, -20 degrees C and -80 degrees C) and analysed at selected times during one year. Determination was performed on the first, third and seventh day during the first week, then once a week for three weeks, once every two weeks for four weeks, then once a month for 4 months and finally, once every 2 months. Extraction was performed using liquid-liquid extraction with 1-chlorobutane, while quantification was carried out using high performance liquid chromatography equipped with a photodiode-array ultraviolet detector. At room temperature, the concentration of all benzodiazepines decreased over one year to 100 and 70% for low and high concentrations, respectively. At 4 degrees C, the decrease was between 90 and 100% for low concentrations and between 50 and 80% for high concentrations. At -20 degrees C, the measured decrease was between 10 and 20% for high and low concentrations, respectively. At -80 degrees C, the measured loss was not significant at high concentration except for midazolam. However, at low concentration the determined decrease was between 5 and 12%. The data collected suggests that quantitative results concerning long-term stored samples should be interpreted with caution in forensic cases. Further investigations concerning the stability of drugs in whole blood or other biological samples, additional methods of identification and determination as well as the establishment of optimal storage conditions should be undertaken in forensic cases.
A HPLC-UV determination of clobazam and N-desmethylclobazam in human serum and urine is presented. After simple liquid-liquid extraction with dichloromethane the compounds and an internal standard diazepam were separated on a Supelcosil LC-8-DB column at ambient temperature under isocratic conditions using the mobile phase: CH3CN-water-0.5 M KH2PO4-H3PO4 (440:540:20:0.4, v/v and 360:580:60:0.4, v/v for serum and urine, respectively). The detection was performed at 228 nm with limits of quantification of 2 ng/ml for serum and 1 ng/ml for urine. Relative standard deviations for intra- and inter-assay precision were found below 8% for both compounds for all the tested concentrations. The described procedure may be easily adapted for several 1,4-benzodiazepines.
The analysis of clobazam by high-performance liquid chromatography and UV detection is described herein. After adding an internal standard, 600 microl of plasma were extracted under basic conditions onto disposable cartridges packed with celite. The organic extract was then evaporated to dryness and the residue reconstituted in 200 microl of mobile phase. A 20 microl aliquot was injected into chromatograph. The HPLC system was equipped with an Ultrasphere C8 analytical column coupled with an UV detector set at 235 nm. The mobile phase was an acetate buffer 20 mM, pH 5.5, containing acetonitrile and triethylamine 70:30:0.01 (v/v); the flow-rate was 1.8 ml/min. Using this method, clobazam can be detected with a sensitivity limit of 6 ng/ml and the RSD% intra- and inter-assay were lower than 5%. For its ruggedness and reliability, the proposed method is particularly suitable for therapeutic drug monitoring in epilepsy.
We report two cases of overdoses of intramuscular midazolam used as a premedication. Both cases had no resedation or complications, but the accidents happened as a result of a resident and nurse's lack of experience with midazolam. The intramuscular doses, given at four times the normal quantity, fortunately caused no harm in our cases. However, the situations suggest that we should carefully check the dosage and review the correct procedures, even when using a drug that is considered to be familiar with most practitioners.
Clobazam (Castillium, Urbanil), a benzodiazepine often used as an anxiolytic and in the treatment of epilepsy, is considered a relatively safe drug. The authors present a fatal case with a 49-year-old female, found dead at home. She had been undergoing psychiatric treatment and was a chronic alcoholic. The autopsy findings were unremarkable, except for multivisceral congestion, steatosis and a small piece of a plastic blister pack in the stomach. Bronchopneumonia, bronchitis and bronchiolitis were also diagnosed. Anhigh-performance liquid chromatography (HPLC)/diode array detector (DAD)/mass spectrometry detection (MSD) with electrospray method was developed in order to detect, confirm and quantify clobazam in the post-mortem samples. In the chromatographic separation, a reversed-phase column C18 (2.1 x 150 mm, 3.5 microm) was used with a mobile phase of methanol and water, at a 0.25 ml/min flow rate. Carbonate buffer (pH 10.5) and 20 microl of prazepam (100 microg/ml) as internal standard were added to the samples. A simple and reliable liquid-liquid extraction method for the determination of clobazam in post-mortem samples was described. Calibration curves for clobazam were performed in blood, achieving linearity between 0.01 and 10 microg/ml and a detection limit of 1.0 ng/ml. The clobazam concentration found in post-mortem blood was 3.9 microg/ml, higher than the reported therapeutic concentration (0.1-0.4 microg/ml). The simultaneous acquisition by photodiode array detection and mass spectrometry detection results allowed benzodiazepines to be identified with sufficient certainty. An examination of all the available information suggested that death resulted from respiratory depression due to clobazam toxicity.
This paper outlines the toxicology results from 1014 cases of claimed drug-facilitated sexual assault (DFSA) analysed at the Forensic Science Service, London Laboratory between January 2000 and December 2002. Where appropriate, either a whole blood sample and/or a urine sample was analysed for alcohol, common drugs of abuse and potentially stupefying drugs. The results were interpreted with respect to the number of drugs detected and an attempt was made to distinguish between voluntary and involuntary ingestion from information supplied. Alcohol (either alone or with an illicit and/or medicinal drug) was detected in 470 of all cases (46%). Illicit drugs were detected in 344 cases (34%), with cannabis being the most commonly detected (26% of cases), followed by cocaine (11%). In 21 cases (2%), a sedative or disinhibiting drug was detected which had not been admitted and could therefore be an instance of deliberate spiking. This included three cases in which complainants were allegedly given Ecstasy (MDMA) without their knowledge. Other drugs detected included gammahydroxybutyrate (GHB) and the benzodiazepine drugs diazepam and temazepam. Another nine cases (1%) involved the complainant being either given or forced to ingest pharmaceutical tablets or an illicit drug.
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