Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

Determination of benzodiazepines in human hair by on-line high-performance liquid chromatography using a restricted access extraction column.

A method is described for the identification of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam and oxazepam) in human hair samples by reversed phase HPLC, following on-line simple enrichment and clean-up on a restricted access extraction column. 50mg of powdered hair were incubated (2h at 45 degrees C) after sonication (1h) in 1 ml of the following solution (methanol:ammonia, 97.5/2.5, v/v). The aliquot was centrifuged and the methanolic phase transferred to a conical tube and evaporated under a gentle stream of nitrogen. The residue was reconstituted by adding 100 microl of a mixture of phosphate buffer (20mM, pH=2.2) and acetonitrile (94/6, v/v). A total of 80 microl were injected into the system with the column switching technique. The pre-column or clean-up column was washed with phosphate buffer pH=7.2. The drugs retained on the pre-column were then eluted in the back-flush mode and separated on a C(8) semi micro column, Lichrospher select B, 125 mm x 3 mm. The BZD were determined by a photodiode-array detector at 254 nm, using reference data (retention time and UV spectra) stored in a personal library. The method showed excellent linearity between 0.5 and 20 ng/mg of hair for clonazepam, flunitrazepam and midazolam and between 0.5 and 100 ng/mg of hair for diazepam and oxazepam. Finally, the present method has been applied to a number of forensic cases in our laboratory.     

超分子溶剂萃取-气相色谱-串联质谱法检测尿液中苯二氮䓬类药物

目的建立一种尿液中9种苯二氮?类药物的超分子溶剂样品气相色谱-串联质谱(gas chromatography-tandem mass spectrometry,GC-MS/MS)分析方法。方法含9种苯二氮?类药物对照品的尿液样品用四氢呋喃和1-己醇组成的超分子溶剂进行液液萃取,取溶剂层氮吹至干,残余物用甲醇复溶后进行GC-MS/MS分析,数据采集方式为多反应监测模式,采用内标法定量。结果尿液中地西泮、咪达唑仑、氟硝西泮和氯氮平质量浓度在1~100ng/mL,劳拉西泮和阿普唑仑质量浓度在5~100ng/mL,硝西泮和氯硝西泮质量浓度在2~100ng/mL,艾司唑仑在质量浓度0.2~100ng/mL范围内具有良好的线性关系,相关系数为0.9991~0.9999,定量下限为0.2~5ng/mL,提取回收率为81.12%~99.52%,日内精密度[相对标准偏差(relative standard deviation,RSD)]和准确度(偏倚)分别小于9.86%、9.51%;日间精密度(RSD)和准确度(偏倚)分别小于8.74%、9.98%。室温和-20℃条件下,尿液中9种药物在15d内具有良好的稳定性。8名志愿者单摄口服阿普唑仑片后,在8~72h内尿液中阿普唑仑的质量浓度为6.54~88.28ng/mL。结论本研究建立的尿液中9种苯二氮?类药物的超分子溶剂萃取-GC-MS/MS分析方法,简便、快速、准确、灵敏,可为临床治疗及司法鉴定中苯二氮?类药物中毒监测提供技术支持。     

Detection of Acute Diazepam Exposure in Bone and Marrow: Influence of Tissue Type and the Dose‐Death Interval on Sensitivity of Detection by ELISA with Liquid Chromatography Tandem Mass Spectrometry Confirmation*

Abstract: Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC/MS/MS) were used to detect diazepam exposure in skeletal tissues of rats (n = 15) given diazepam acutely (20 mg/kg, i.p.), and killed at various times postdose. Marrow, epiphyseal, and diaphyseal bone were isolated from extracted femora. Bone was cleaned, ground, and incubated in methanol. Marrow underwent ultrasonic homogenization. Extracts and homogenates were diluted in phosphate buffer, and then underwent solid‐phase extraction and ELISA. Relative sensitivity of detection was examined in terms of relative decrease in absorbance (ELISA) and binary classification sensitivity (ELISA and LC/MS/MS). Overall, the data showed differences in relative sensitivity of detection of diazepam exposure in different tissue types (marrow > epiphyseal bone > diaphyseal bone), which is suggestive of heterogenous distribution in these tissues, and a decreasing sensitivity with increasing dose‐death interval. Thus, the tissue type sampled and dose‐death interval may contribute to the probability of detection of diazepam exposure in skeletal tissues.     

Detection of drugs in saliva of impaired drivers

This study examined the feasibility of detecting drugs using saliva samples obtained from impaired drivers. Screening procedures on 1- to 2-mL samples were for cannabinoids, volatiles, benzodiazepines, and other acidic/neutral/basic drugs. Methodology consisted of enzyme multiple immunoassay technique (EMIT) and temperature programmed gas chromatography with confirmation by gas chromatography/mass spectrometry (GC/MS). Fifty-six samples were obtained from drivers arrested for suspicion of impaired driving. Other than alcohol, the major drugs detected were cannabinoids and diazepam. Cocaine was found in one case.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.     

三种安定类药物在中毒家兔体内的分布研究

为探求安定类药物在急性中毒家兔体内的代谢分布，采用固相萃取法和HPLC同时测定氯硝安定、佳静安定和安定。家兔按一定剂量将三种药物同时灌胃造成急性中毒2小时，处死后测定血、胆汁、尿液及其它脏器的三种药物浓度。结果表明，氯硝安定在胆汁和尿中含量较高，为0．82μg／ml和0．68μg／ml，而佳静安定及安定在胆汁和肝中浓度高，分别为9．32μg／ml和13．81μg／g。提示三种药物在家兔体内分布既有相同点也有显著差别。因此，利用本方法既可同时测定三种不同药物，也可为法医检案中毒案例的最佳检材采取和毒物分析结果评价提供了科学依据。     

A study into the rate of incorporation of eight benzodiazepines into rat hair

The incorporation of eight benzodiazepines (chlordiazepoxide, diazepam, estazolam, flunitrazepam, flurazepam, medazepam, oxazepam and triazolam) into rat hair was investigated by HPLC and GC-MS. Each of the benzodiazepines was injected daily into three Dark Agouti (DA) rats for 10 days at 10mg/kg. The back hair of the rats was removed by shaving prior to the first injection and again on the 28th day after the initial administration.To investigate optimum extraction conditions, 10mg aliquots of rat hair incorporated with diazepam, flurazepam or medazepam were extracted by seven different methods (Proteinase K, methanol-ammonia, methanol-trifluoroacetic acid, Soerensens buffer, 1M NaOH, beta-glucuronidase/arylsulfatase, Biopurase). The method found to yield the highest recoveries, for all three drugs, was the acidic methanol extraction. Using this extraction procedure, the incorporation rates (ICR: the ratio of the hair concentration to the plasma AUC) of eight benzodiazepines into rat hair were investigated. The ICRs ranged from 0.002 (flunitrazepam) to 0.049 (flurazepam).The major metabolites of flurazepam were investigated in rat hair. The mean hair concentrations of desalkylflurazepam and 2-hydroxyethylflurazepam were 3.31 and 0.05 ng/mg, respectively, which are 24 and 0.36% of the parent compound in hair.     

新型微流体液相色谱(micro-LC)串联四极杆质谱检测头发中5种苯二氮卓类药物

目的建立了新型微流体液相色谱(micro-LC)串联四级杆质谱检测头发中5种苯二氮卓类药物(三唑仑,α-羟基咪达唑仑,咪达唑仑,α-羟基阿普唑仑,普拉西泮)。方法采用液-液萃取法(LLE)提取毛发样品,微流体液相色谱串联四级杆质谱检测。结果在回归系数大于0.99的情况下,目标分析物的良好线性。精密度和萃取效率分别在2.7%~17.1%和66.5%~120.5%之间。每个分析物的检出限和定量限分别为0.001~0.08 pg·mg^-1和0.0125~0.25pg·mg^-1。结论本方法与传统的液相色谱串联质谱法(LC/MS-MS)相比灵敏度出高2-10倍。本研究展示了新型微流体分离系统串联质谱方法的实用性,并为法医毒理学领域关于头发样本中痕量药物检测提供了一种新的检测手段。     

Detection and validated quantification of 21 benzodiazepines and 3 "z-drugs" in human hair by LC-MS/MS

A method for detection and quantification of 21 benzodiazepines and the pharmacologically related "z-drugs" in human hair samples was developed and fully validated using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After methanolic and methanolic/aqueous extraction, the analytes were separated using two different LC-MS systems (AB Sciex 3200 QTRAP and AB Sciex 5500 QTRAP). Separation columns, mobile phases and MS modes for both systems were: Phenomenex Kinetex, 2.6 μm, 50/2.1; 5mM ammonium formate buffer pH 3.5/methanol, total flow 0.75 mL/min; electrospray ionization (ESI), multiple reaction monitoring (MRM), information dependent acquisition (IDA), enhanced product ion scan (EPI). The assays were found to be selective for the tested compounds (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxymidazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem and zopiclone), all validation criteria were in the required ranges according to international guidelines, except for bromazepam. Matrix effects, and process efficiencies were in the acceptable ranges evaluated using the post-extraction addition approach. Lower limits of quantification were between 0.6 and 16 pg/mg of hair. The LC-MS/MS assay has proven to be applicable for determination of the studied analytes in human hair in numerous authentic cases (n=175).     

7-[125I]iodoclonazepam: purification by high-performance liquid chromatography for use in a very sensitive benzodiazepine radioimmunoassay of broad specificity

The purification of 7-[125I]iodoclonazepam by high-performance liquid chromatography (HPLC) for use in a very sensitive benzodiazepine radioimmunoassay is described. A silica column is used with a non-aqueous eluent and sequential ultra-violet and gamma-ray detection. A commercially available antiserum is used at a dilution of 1:1000. Blood samples are diluted 10-fold with buffer before analysis and only 25 microliters of diluted sample are required per assay tube. Benzodiazepines, but not the radiolabel, appear to be bound by blood proteins in competition with the antiserum and so, if undiluted blood is assayed, erroneously low results are obtained. The minimal sample requirement and the high sensitivity of the assay described here largely avoid this problem while maintaining acceptable detection limits. For diazepam, the detection limit is 2.5 ng/ml in blood or urine (after correction for the initial 10-fold dilution) and therapeutic or sub-therapeutic levels of many other benzodiazepines can be detected. In practice, the assay is reliable, simple to perform and extremely economical.     

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.     

Methadone‐Related Deaths – Epidemiological,Pathohistological, and Toxicological Traits in 10‐year Retrospective Study in Vojvodina,Serbia

The number of methadone‐related deaths (MRDs) during a 10‐year period (2002–2011) in the region of Vojvodina, Serbia, was increased. The cases were evaluated according to epidemiological parameters, pathohistological findings, and toxicological screening. The majority of victims were men, aged from 20 to 38. Pathohistologically, the signs of acute focal myocardial damage were present in the heart of victims with drug abuse history shorter than 2 years, while both signs of recent and chronic focal myocardial damage were developed among victims with longer drug abuse history (2–5 years). In postmortem blood samples of 54.84% of victims, methadone was detected in combination with diazepam, both in therapeutic range. Alcohol was absent in most cases. Other detected drugs were antipsychotics and antidepressants in therapeutic concentrations. These findings raise the attention to the concomitant use of methadone and benzodiazepines with the need for further studies to clarify the mechanism of death in such cases.     

A survey of buprenorphine related deaths in Singapore

Buprenorphine is available in Singapore as substitution treatment for opioid dependence since 2002. This study surveys buprenorphine related deaths in Singapore between September 2003 and December 2004. The aims are to establish the autopsy prevalence of buprenorphine related deaths and the demographical and toxicological profile of the cases. Toxicological screening was performed for all unnatural deaths, deaths involving known drug addicts, as well as when autopsy revealed no obvious cause of death. Twenty-one cases had buprenorphine detected in post-mortem blood and/or urine samples. Eighteen were sudden deaths. There were two fatal falls from height and one death by hanging. All subjects were male. The age range was 24-48 years. Fourteen subjects were between 30 and 39 years of age. The mean age was 35 years. The majority (62%) were Chinese. Eleven (52%) were known drug abusers. For sudden deaths, two groups were identified. Six cases died from natural causes. Blood buprenorphine levels ranged from undetected (detected in urine) to 3.2 ng/mL (mean 1.4 ng/mL). Twelve cases were attributed directly and indirectly to mixed drug poisoning. Blood buprenorphine levels ranged from undetected (detected in urine) to 17 ng/mL (mean 3.2 ng/mL). Nineteen cases showed concurrent abuse of buprenorphine and benzodiazepine, diazepam being the most frequently detected, followed by nitrazepam and midazolam. The availability of buprenorphine as substitution therapy is associated with an increase in buprenorphine related deaths. The danger of co-abuse of buprenorphine and benzodiazepines is highlighted.     

The detection of sedatives in hair and nail samples using tandem LC-MS-MS

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.     

Quantitation using GC-TOF-MS: example of bromazepam

Time-of-flight mass spectrometry (TOF-MS) offers new perspectives for forensic toxicology. Qualitative and quantitative analyses of a mixture of three selected benzodiazepines (diazepam, nordazepam and bromazepam) were used to compare gas chromatography (GC-TOF-MS, quadrupole GC-MS, GC-ECD) and liquid chromatography (HPLC-DAD) data. Method validation parameters like LOD, LOQ, S/N-ratios reflect the capabilities of GC-TOF-MS. Five-point calibrations for bromazepam in human peripheral blood (50, 100, 160, 200, 300 ng/ml) using medazepam as internal standard (1000 ng/ml) were performed. The calibrations using GC-TOF-MS (using the fragments of m/z 236 and 288), GC-ECD (dual system) and HPLC-DAD (at 235 nm) all showed correlation coefficients close or superior to 0.99. Quadrupole GC-MS data was not used in the comparison of extracted samples due to the low sensitivity in the full scan mode. Two analyses of real cases concerning bromazepam are presented. In the first case, the presence or absence of bromazepam could not be established with both HPLC-DAD and GC-ECD due to background signals. The extracted ion chromatograms and spectrum traces after the analysis with the GC-TOF-MS could clearly excluded the presence of bromazepam. The second case illustrates the quantitation of bromazepam, where both HPLC-DAD and GC-ECD were unable to give satisfactory results, again due to interfering background signals. The analyses performed on the GC-TOF-MS-system demonstrated high sensitivity and also high selectivity due to the high quality of mass spectra obtained. The advantages of GC-TOF-MS make it a promising analytical technique for forensic toxicology.     

Influence of bleaching on stability of benzodiazepines in hair

In order to study the influence of hair bleaching on benzodiazepines concentrations, hair was treated with a bleaching product (Poly Blonde, Schwarzkopf & Henkel) for 20 min. The treated hair specimen was obtained from a person who died after an overdose of several illicit drugs associated with benzodiazepines. Bleached and non bleached hair were washed (acetone and water), pulverised and then incubated for 2 h in a thioglycolic solution. In the extracts obtained by solid-phase extraction on C18 columns, the different drugs with the corresponding deuterated standards were derivatized and determined by GC-MS in a SIM mode. These results show that the concentrations of all the drug detected decreased in bleached hair in comparison with non treated hair. Whereas the diminution was less important for cocaine and benzoylecgonine (decrease of 24.6 and 36.4%, respectively), concentrations for codeine, 6-monoacetylmorphine and morphine decreased more significantly (decrease of 57.5, 88.6 and 67.4%, respectively) as well as those of diazepam, nordazepam and 7-aminoflunitrazepam (decrease of 39.7, 67.7 and 61.8%, respectively). The results in this study agree with those of other authors that bleaching affects the stability of cocaine and opiates incorporated in hair. These findings also point out that bleaching influences the stability of entrapped benzodiazepines in hair. Finally, these results reconfirm that it is very important to consider the cosmetic history of a hair sample in the interpretation of hair analysis results.     

Drug screening and confirmation by GC-MS: comparison of EMIT II and Online KIMS against 10 drugs between US and England laboratories

Drug screening through urinalysis is a widely accepted tool for rapid detection of potential drug use at a relatively low cost. It is, therefore, a potentially useful method for detecting and monitoring drug use in a variety of contexts such as the criminal justice system, pre-employment screening and a variety of treatment centers. This article explores the efficacy of two commercially available drug-screening assays: Online KIMS assay (Roche) and EMIT II assays. First, we evaluate the sensitivity and specificity of two immunoassays. A total of 738 urine samples were collected among adult arrestee populations from Chicago, New Orleans and Seattle through the Arrestee Drug Abuse Monitoring (ADAM) program. Partial samples were split within one laboratory and analyzed by both enzymes multiplied immunoassay technique (EMIT) II and kinetic interaction of microparticle in solution (KIMS) assays for a 10-drug panel (amphetamine, barbiturates, benzodiazepines, marijuana, cocaine, methadone, methaqualone, opiate, phencyclidine and propoxyphene). Gas chromatography-mass spectrometry (GC-MS) was used as a confirmation method for all positives from either EMIT II or KIMS for all experiments. Second, the paper examines whether using different testing laboratories plays a role in the final results. The same experiments were repeated at two different testing locations: one in California and one in London and England. Third, the paper studies whether drug testing results vary between two laboratories when each of them had used their own routine screening method: the Forensic Science Service (FSS) at Birmingham, United Kingdom with KIMS assay and Medscreen Limited at London, United Kingdom with EMIT II. In summary, both EMIT II and KIMS assays generate fairly consistent results. The concordance rate against each of the 10 drugs tested is relatively high (97.4-100%). The discrepancies, in most cases, occurred at drug concentrations near the cut-off levels. There were more discrepant results between two laboratories compared to when specimens were analyzed at the same laboratory using two different assays.     

A screening radioimmunoassay for 1,4-benzodiazepines in human blood, serum, and urine using anti-oxazepam-hemisuccinate-antibodies (author's transl)]

A simple and specific radioimmunoassay (RIA) was developed for the determination of oxazepam and other 1,4-benzodiazepines in human blood serum and urine (e.g., diazepam, desmethyldiazepam, chlorazepate). For serum a 1:10 dilution, for urine a 1:100 dilution is recommended. Blood and hemolyzed samples need prior extraction by Amberlite XAD-2. The antisera were raised by immunizing "White New Zealand"-rabbits with an oxazepam-3-hemisuccinate bovine serum albumin conjugate. Using 0.1 ml serum dilution the sensitivity is 0.01 mg/l per tube. Especially higher concentrations show a tendency toward underestimation. Being not limited to a single 1,4-benzodiazepine derivative, the specificity of the antisera is also suitable for a screening analysis. Compared to thin-layer chromatographic analysis of urine this assay shows improved sensitivity (0.05--0.1 mg/l in 0.1 ml of a 1:100 dilution = 1 microliter of urine). For forensic investigations, an analysis in the sequence of urine-RIA, blood/serum-RIA, blood/serum-"electron-capture"-gas-liquid chromatography (ECD-GLC) seems to be a helpful approach. Blood levels of diazepam and desmethyldiazepam determined by RIA and GLC after extraction are in satisfactory agreement.