MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats.

The underlying mechanisms of cardiotoxicity of 3,4-methylenedioxymethylamphetamine (MDMA, "ecstasy") abuse are unclear. Autophagy exerts either adaptive or maladaptive effects on cardiac function in various pathological settings, but nothing is known on the role of autophagy in the MDMA cardiotoxicity. Here, we investigated the mechanism through which autophagy may be involved in MDMA-induced cardiac contractile dysfunction. Rats were injected intraperitoneally with MDMA (20mg/kg) or saline. Left ventricular (LV) echocardiography and LV pressure measurement demonstrated reduction of LV systolic contractility 24h after MDMA administration. Western blot analysis showed a time-dependent increase in the levels of microtubule-associated protein light chain 3-II (LC3-II) and cathepsin-D after MDMA administration. Electron microscopy showed the presence of autophagic vacuoles in cardiomyocytes. MDMA upregulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172, mammalian target of rapamycin (mTOR) at Thr2446, Raptor at Ser792, and Unc51-like kinase (ULK1) at Ser555, suggesting activation of autophagy through the AMPK-mTOR pathway. The effects of autophagic inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) on LC3-II levels indicated that MDMA enhanced autophagosome formation, but attenuated autophagosome clearance. MDMA also induced release of cathepsins into cytosol, and western blotting and electron microscopy showed cardiac troponin I (cTnI) degradation and myofibril damage, respectively. 3-MA, CQ, and a lysosomal inhibitor, E64c, inhibited cTnI proteolysis and improved contractile dysfunction after MDMA administration. In conclusion, MDMA causes lysosome destabilization following activation of the autophagy-lysosomal pathway, through which released lysosomal proteases damage myofibrils and induce LV systolic dysfunction in rat heart.

Benzalkonium chloride intoxication caused by intravenous self-injection.

BACKGROUND: Benzalkonium chloride (BZK) is widely used as a germicide in hospitals and other places. Although several cases of accidental oral intake of BZK have been reported, there have been few reported cases of BZK toxicity due to intravenous injection. CASE REPORT: A male nurse in his 40 s injected 15 mL of 10% BZK (Osvan S) directly into his left antebrachial vein while at home, as a suicide attempt. The patient was admitted to our hospital 1 hour later. Acute respiratory distress syndrome (ARDS) was diagnosed by blood gas analysis, chest X-ray, and CT scan. Due to extracorporeal blood purification therapy, including hemoperfusion and plasma exchange, serum BZK became undetectable. However, the ARDS was not improved. Extracorporeal blood purification therapy consisting of continuous hemodiafiltration (CHDF) was continued to treat the ARDS. After performing CHDF for the next 36 hours, improvement of both the PaO2/FiO2 ratio and chest X-ray findings was noted. Tracheal extubation was performed on day 9 and no further complications occurred after this period, he was discharged on day 21. CONCLUSION: Extracorporeal blood purification therapy is probably effective for treatment of BZK intoxication by intravenous injection.

Relationship between the matrix effect and the physicochemical properties of analytes in gas chromatography.

The phenomenon "matrix-induced chromatographic response enhancement" (matrix effect) causes quantitative errors in gas chromatography (GC) analyses. This effect varies according to the analyte nature, matrix type and concentration, and GC-system parameters. By focusing on the physicochemical properties of analytes, a predictive model was developed for the matrix effect using quantitative structure-property relationships. Experimental values of the matrix effect were determined for 58 compounds in a serum extract obtained from solid-phase extraction as the matrix. Eight molecular descriptors were selected, and the matrix-effect model was developed by multiple linear regression. The developed model predicted values for the matrix effect without any further experimental measurements. It also indicated that the molecular polarity (particularly H-bond donors) and volume of the analyte increase the matrix effect, while hydrophobicity and increasing number of nonpolar carbon atoms in the analyte decrease the matrix effect. The model was applied to the analysis of barbiturates. The predicted values indicated that N-methylation decreases the matrix effect, and the relative predicted values were effective for the selection of an internal standard. The obtained insight into the matrix effect and the prediction data will be helpful for developing quantitative analysis strategies.

Abdominal Stab Wounds with Tension Pneumopericardium Confirmed by Autopsy and Postmortem Computed Tomography.

We present the first report of pneumopericardium observed by autopsy and on postmortem computed tomography (PMCT) images. The subject was a woman who died of self-inflicted stab wounds to the abdomen. The PMCT scan revealed air in the pericardial sac, a "flattened heart" sign, and retroperitoneal hemorrhage. Medicolegal autopsy revealed two abdominal stab wounds near the xiphoid process that had cut the apical pericardium and adjacent diaphragm and liver. Examination of the open thorax confirmed that the pericardial sac was distended with air. The wound extended to the abdominal aorta, causing retroperitoneal hemorrhage. PMCT images showed that the pneumopericardial volume was 133 mL. We believe that cardiac tamponade occurred resulting from the tension pneumopericardium; however, the effects were mitigated by hypovolemia secondary to the retroperitoneal hemorrhage as well as obstructive shock. Therefore, the cause of death appears to have been low-pressure cardiac tamponade.

Miniaturized sample preparation needle: a versatile design for the rapid analysis of smoking-related compounds in hair and air samples.

Miniaturized needle extraction device has been developed as a versatile sample preparation device designed for the rapid and simple analysis of smoking-related compounds in smokers' hair samples and environmental tobacco smoke. Packed with polymeric particle, the resulting particle-packed needle was employed as a miniaturized sample preparation device for the analysis of typical volatile organic compounds in tobacco smoke. Introducing a bundle of polymer-coated filaments as the extraction medium, the needle was further applied as a novel sample preparation device containing simultaneous derivatization/extraction process of volatile aldehydes. Formaldehyde (FA) and acetaldehyde (AA) in smoker's breath during the smoking were successfully derivatized with two derivatization reagents in the polymer-coated fiber-packed needle device followed by the separation and determination in gas chromatography (GC). Smokers' hair samples were also packed into the needle, allowing the direct extraction of nicotine from the hair sample in a conventional GC injector. Optimizing the main experimental parameters for each technique, successful determination of several smoking-related compounds with these needle extraction methods has been demonstrated.

Effects of long-term administrations of aconitine on electrocardiogram and tissue concentrations of aconitine and its metabolites in mice.

Aconitum alkaloids are well known for their acute and high toxicity, for example, in the causation of severe arrhythmias leading to death. Aconitine, one of the major Aconitum alkaloids, is a highly toxic compound from the Aconitum species. However, there has been no studies reported on the influence of the chronic administration of aconitine. Thus, this study was conducted to investigate the influence of chronic administration of aconitine in experimental animal models. A dose of 1mg/kg per day was administered to the experimental animal models. We determined the concentration of aconitine and its metabolites (benzoylaconine and aconine) in organs and blood with gas chromatography/selected ion monitoring (GC/SIM). In addition, we concurrently recorded the electrocardiogram (ECG). Fifteen minutes after administration on day 0, the early aconitine administered group (acute group) revealed peak organ and blood concentration levels of aconitine with a gradual decrease, thereafter. The concentration of aconitine in organs and blood (from days 0 to 22; 90 min after the last administration of aconitine) gradually decreased according to repeated administration, whereas benzoylaconine and aconine increased. ECG revealed various types of arrhythmias. However, the frequency of arrhythmias remarkably decreased with time and repeated administration of aconitine. These results indicate two possibilities. First, the increase in the activity of aconitine metabolism. Secondly, the decrease of effectiveness to the heart due to long-term (chronic) administration of aconitine.

Diagnostic performance of Triage for benzodiazepines: urine analysis of the dose of therapeutic cases.

We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given therapeutic doses of benzodiazepines and compared the results of EMIT assays. Results obtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assays was 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assays were different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of thienodiazepines.

Evaluation of residual toxic substances in the stomach using upper gastrointestinal endoscopy for management of patients with oral drug overdose on admission: a prospective, observational study.

The guidelines on the indications for gastric lavage were published in 1997, and a less-aggressive initial approach has been used for poisoned patients. Clinical studies have shown that the outcomes of retrieval of residual toxic substances in the stomach are variable and that no beneficial effect is obtained. However, the presence of residual toxic substances in the stomach before gastric lavage has not been estimated. The objective of this study was to evaluate the residual stomach contents on admission of patients with oral drug overdoses using upper gastrointestinal endoscopy. A 2-year prospective study of 167 patients with oral drug overdoses was performed. Endoscopy was performed on admission to observe the gastric body, fornix, and pyloric antrum. Patients were classified into 3 groups according to the digestive phase (tablet/food phase, soluble/fluid phase, and reticular/empty phase). The groups were compared with respect to time elapsed since ingestion, and numbers and variety of orally overdosed drugs. The numbers of patients in each phase were as follows: tablet/food phase, 73; soluble/fluid phase, 50; and reticular/empty phase, 44. The tablet/food and soluble/fluid phase groups contained the greatest numbers of patients who presented within 1 to 2 hours since ingestion. In the tablet/food group, only 12 of 73 patients (16%) presented within 1 hour since ingestion, and 3 patients presented >12 hours since ingestion. In the soluble/fluid phase group, only 9 of 50 patients (18%) presented within 1 hour since ingestion, and 2 patients presented >12 hours since ingestion. The reticular/empty phase group contained the greatest number of patients presenting within 2 to 4 hours since ingestion, and 3 patients presented within 1 hour since ingestion. The residual stomach contents before lavage were variable in all of the groups. The residual gastric content before the performance of gastric lavage is variable in overdosed patients on admission. This may influence the efficiency of gastric lavage with respect to retrieval of residual toxic substances in the stomach. This study may contribute to the development of a strategy for treating patients who have orally overdosed on drugs in the future.

Forensic toxicological analyses of drugs in tissues in formalin solutions and in fixatives.

Forensic toxicological drug analyses of human specimens are usually performed immediately after autopsy or on frozen preserved tissues. Occasionally, cases require analysis of drugs from tissues fixed in formalin solution. To improve the estimation of the level of drug in tissues following formalin fixation, we studied drug concentrations in human tissues, liver and kidney, that were collected from a drug-positive autopsy case. Parts of tissues were preserved in formalin solution for 1, 3, 6 and 13 months. Tissues obtained before and after preservation, along with tissue-exposed fixatives, were assayed using gas chromatography-mass spectrometry; all of the samples were assayed for the presence of drugs and changes in the drug concentrations both before and after preservation in formalin. Concentrations of assayed drugs decreased upon fixation in formalin; levels of these drugs did not necessarily show further decreases during subsequent storage in fixative, up to 13 months. Distinct trends in drug levels were found in liver and kidney. In liver, the levels of chlorpromazine, levomepromazine, and promethazine decreased to 23-39% at 1 month after preservation; all 3 of these drugs were detected at all tested time points of preservation. Bromazepam was not detected at 13 months after preservation. Milnacipran was the most unstable after preservation in formalin solution among all of the assayed drugs. In kidney, all assayed drugs exhibited reduced stability during preservation compared to levels in liver. Methamphetamine and methylenedioxymethamphetamine were not detected in any time points of tissues. The proportions of the drugs that remained within the tissues differed between liver and kidney. Also, S-oxide compounds of chlorpromazine and levomepromazine, which were not observed before preservation, were detected in fixed liver tissues and their fixatives at 3, 6 and 13 months of preservation. These results suggest that analyses in formalin-fixed tissues need to include analysis of various organ-tissues and their fixatives at multiple time points for the duration of preservation. These analyses should include detection of chemical degradation/denaturation products, such as S-oxides of chlorpromazine and levomepromazine.

Polymer-coated fibrous extraction medium for sample preparation coupled to microcolumn liquid-phase separations.

Polymer-coated fibrous material has been introduced as the extraction medium for a miniaturized sample preparation method being coupled with microcolumn liquid chromatography. The preconcentration and the subsequent liquid chromatographic separation of tricyclic antidepressants (TCAs) drugs, amitriptyline, imipramine, nortriptyline and desipramine, was carried out with the hyphenated system. Several basic experimental parameters, such as extraction and separation conditions, were investigated along with the applicability of the method for the analysis of biological fluids. The results clearly showed that the on-line coupled system could be a powerful tool for the analysis of complex mixtures in biological matrix without a large solvent consumption and specially designed instruments. The lowest limit of quantification was quite acceptable for the analysis of TCAs in clinical and forensic situations.

Sensitive determination of tetrodotoxin using column-switching liquid chromatography-mass spectrometry with electrospray ionization in mouse serum.

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method coupled with a column-switching technique was developed for the determination of tetrodotoxin (TTX) in serum. An on-column, column-switching technique was employed to analyze TTX without pretreatment of the serum. The combination of a multimode column with reversed phases and cation exchange for TTX provided successful separation and MS determination in the ESI-positive mode. A 100-microL serum sample was injected directly into a precolumn. For TTX monitored at m/z 320.1 in the selective ion monitoring mode, the calibration curve was linear within the range 0.1-100 ng/mL, and the limit of detection was 0.5 ng/mL. Recoveries were around 90% for TTX. The present method allows successful analysis of TTX in serum. In conclusion, this new method is simple, accurate, and useful for the determination of TTX and should be of benefit to both forensic and clinical toxicology.

Miniaturized sample preparation and separation methods for environmental and drug analyses.

Miniaturized extraction and separation media have been successfully developed from precisely controlled technologies. In this article, recent developments in these high performance analytical methods, such as miniaturized sample preparation methods and the coupling of these techniques with microscale separation systems, have been reviewed, along with some applications to environmental and biological analysis. The advantage of the miniaturization is not only for the environmental compatibility but also for the developments of the high performance analytical systems. Down-sizing also makes it possible to investigate and introduce various compounds and materials as novel media (such as tailor-made materials and devices) in separation science. As a typical example of the novel miniaturized sample preparation system, the applications of fibrous materials for microcolumn liquid-phase separation methods are described.

A column-switching LC/MS/ESI method for detecting tetrodotoxin and Aconitum alkaloids in serum.

A liquid chromatography-mass spectrometry-electrospray ionization (LC/MS/ESI) method coupled with a column-switching technique has been developed for the determination of tetrodotoxin (TTX) and Aconitum alkaloids and their metabolites, such as aconitine, mesaconitine, hypaconitine, jesaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, in serum. An on-column column-switching technique was employed to analyze TTX and Aconitum alkaloids and their metabolites without pretreatment of the serum. Combination of a multimode column with reversed phases and cation exchange for TTX, and use of a multimode column with reversed phases and a hydrophobic polymer column for Aconitum alkaloids and their metabolites provided successful separation and MS determination in ESI positive mode. A 100 microl serum sample was directly injected into a precolumn. For TTX monitored at m/z 320.1 in the selected ion monitoring mode, the calibration curve was linear within the range 0.1-100 ng/ml and the limit of detection was 0.1 ng/ml. For aconitine, mesaconitine, hypaconitine and jesaconitine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 0.2 to 1 ng/ml. For benzoylaconine, benzoylmesaconine, benzoylhypaconine and 14-anisoylaconine, linear calibration curves were obtained up to 500 ng/ml and the limit of detection ranged from 2 to 50 ng/ml. Recoveries from serum samples were within the range 78-119% for all the compounds studied.

[GC-MS analysis of methamphetamine and amphetamine in hair of Thai drug addicts].

UNLABELLED: Psycho-stimulant dependence in young individuals has become a serious problem in Thailand, where consumption of the so-called YaBa methamphetamine tablets has become a fashionable trend. Due to its easy availability in the form of a tablet, young individuals abuse methamphetamine. Methamphetamine tablets are known to be potently addictive and its difficulty in cessation of drug use and to be abstinent from the drug. We herein report the results obtained from GC-MS analysis of methamphetamine and amphetamine in 33 samples of urine and hair from patients with psycho-stimulant dependence. These samples were collected from patients registered at the outpatient clinic in the Department of Psychiatry, Chiang Mai University Hospital and were sent to Nippon Medical School, Department of Legal Medicine (Tokyo, Japan) for further analysis by Dr. Werawan Ruangyttikarn, Department of Forensic Medicine, Chiang Mai University. Sample preparation: Hairs samples were cropped near the hair root. After washing, they were cut into 1.2-cm sections and extracted with methanol/5N HCl (2:1) for an hour and then, solid-phase extraction was conducted using Bond-Elut Certify. Following extraction, GC-MS analysis was performed. Urine samples were subjected to GC-MS analysis after preparation with Bond Elut Certify. RESULTS AND DISCUSSION: In 6 samples, both urine and hair samples analyzed were negative for detection of the stimulant drugs. In those cases individuals might stop taking drug for about 5 months. In 18 samples, urine samples were negative whereas hair samples were positive. These results suggest that individuals might stop using drugs for a few days before they went to the hospital but they abuse drugs continuously. In 9 samples, both urine and hair samples were positive. These results show that individuals always abuse drugs. In order to treat drug dependence effectively it is necessary to obtain the patient history of drug use and to evaluate and determine short-term and long-term drug use with urinalysis and hair analysis, respectively. Our present data revealed that useful information concerned with the long term drug abuse can be obtained from hair analysis, and that this method of analysis is applicable not only to forensic cases but also for evaluating clinical cases.

[Analysis of MDMA and PCP by GC-MS from patients admitted to the critical care medical center].

In our country, abuse of methamphetamine has increased. Furthermore, dealings of other drugs by using internet have increased. But, the poison cases of 3,4-methylenedioxymethamphetamine (MDMA) and phencyclidine (PCP) have never been reported in our country. We report an MDMA poison case and a PCP poison case. We could detect MDMA, MDA or PCP by GC-MS from urine and serum of patients admitted to the critical care medical center of Nippon Medical School. Case 1: A 23-year-old foreign female was admitted to our hospital because of disturbance of consciousness. Her friend said that she had been found lying on the floor of the bathroom after taking a tablet. The screening test by Triage showed AMP positive. Not methamphetamine but MDMA and MDA were detected from urine and serum of the patient by GC-MS. Case 2: A 27-year-old foreign female was admitted to our hospital because of restlessness and excitement. Her friend said that she had become restless and excited after taking 15-30 tablets of Tylenol. The screening by Triage showed BZO and PCP positive. Not acetaminophen but PCP was detected in the patient's sample by GC-MS. Drug abuse has expanded to Japan over the border. New responses to abuse drugs with respect to medical treatment and drug analyses should be established.

Successful retrieval using ultrathin transnasal esophagogastroduodenoscopy of a significant amount of residual tricyclic antidepressant following serious toxicity: a case report.

BACKGROUND: In Japan, ultrathin transnasal esophagogastroduodenoscopy (EGD) with a 4.9-mm diameter endoscope (Olympus XP260) is routinely used to examine the upper gastrointestinal tract. This procedure does not require sedation and does not affect vital signs. Gastric lavage is not empirically employed in the management of all poisoning patients. It is considered only for potentially life-threatening overdoses when the procedure can be performed within 1 h of ingestion of the poison. However, there are no absolute indications for gastric lavage. EGD may increase the indications, efficiency and safety of gastric lavage in poisoning patients. FINDINGS: A 35-year-old female was admitted to our emergency department 2 h after ingesting multiple drugs, including a critical dose of the tricyclic antidepressant (TCA) amitriptyline, at which time she was confused and had a Glasgow Coma Scale score of 8 (E1V2M5). Endotracheal intubation was performed. To confirm the type of TCA and in order to determine whether gastric lavage was required, we decided to perform EGD. Endoscopy demonstrated adherence of residual drugs to the stomach wall, in a soluble form and not as a mass. Hence, gastric lavage was performed via the EGD to avoid passage of these drugs into the small bowel. The patient was extubated on day 2, without the development of complications such as aspiration pneumonia, and was discharged on day 5. CONCLUSION: EGD may be useful in poisoning patients for determining the amount of residual drug in the stomach, also allowing direct observation of the effectiveness of gastric lavage.

Gastric lavage guided by ultrathin transnasal esophagogastroduodenoscopy in a life-threatening case of tobacco extract poisoning: a case report.

Nicotine, which is found in tobacco, is one of the most toxic of all known poisons. A 31-year-old woman was brought to our emergency department 2 hours after ingesting a usually fatal dose of a tobacco extract. Although gastric lavage was once commonly used to treat poisoning cases of this type, lavage can lead to such complications as aspiration, hypoxia, oropharyngeal and gastric trauma, and electrolyte disturbances. Recent guidelines have suggested less-aggressive gastric-emptying procedures as initial treatments. Currently, there are no absolute indications for gastric lavage use. The present patient had a history of depressive episodes and had attempted suicide by ingesting an extract derived from 20 cigarettes mixed with alcohol. There was no evidence of vomiting or seizures occurring before arrival of the ambulance. Physical examination revealed no signs of sweating, although the patient appeared to be confused, which is often seen with nicotine intoxication. She admitted using 2 kinds of cigarette, each of which typically contain 11.2 mg of nicotine. The patient's nicotine level was suspected to be higher than 40 to 60 mg, which is normally fatal. To determine whether gastric lavage was indicated in this case, we performed ultrathin transnasal esophagogastroduodenoscopy, which neither requires sedation nor compromises the airways. Although 2 hours had passed since ingestion, the tobacco extract and food were directly visualized within the stomach. Subsequently, gastric lavage was performed with 2,000 mL of water administered through a nasogastric tube. Ultrathin esophagogastroduodenoscopy made it possible to directly observe the gastric lavage and to ensure the stomach contents had been completely removed. No changes were noted in the vital signs, and no obstruction of the airways was observed. The patient recovered quickly and was discharged the following day. Ultrathin esophagogastroduodenoscopy helped determine the diagnosis and ensure that gastric lavage had been performed without complications.

Antagonistic effects of tetrodotoxin on aconitine-induced cardiac toxicity.

Aconitine, well-known for its high cardiotoxicity, causes severe arrhythmias, such as ventricular tachycardia and ventricular fibrillation, by opening membrane sodium channels. Tetrodotoxin, a membrane sodium-channel blocker, is thought to antagonize aconitine activity. Tetrodotoxin is a potent blocker of the skeletal muscle sodium-channel isoform Na(v)1.4 (IC50 10 nM), but micromolar concentrations of tetrodotoxin are required to inhibit the primary cardiac isoform Na(v)1.5. This suggests that substantial concentrations of tetrodotoxin are required to alleviate the cardiac toxicity caused by aconitine. To elucidate the interaction between aconitine and tetrodotoxin in the cardiovascular and respiratory systems, mixtures of aconitine and tetrodotoxin were simultaneously administered to mice, and the effects on electrocardiograms, breathing rates, and arterial oxygen saturation were examined. Compared with mice treated with aconitine alone, some mice treated with aconitine-tetrodotoxin mixtures showed lower mortality rates and delayed appearance of arrhythmia. The decreased breathing rates and arterial oxygen saturation observed in mice receiving aconitine alone were alleviated in mice that survived after receiving the aconitine-tetrodotoxin mixture; this result suggests that tetrodotoxin is antagonistic to aconitine. When the tetrodotoxin dose is greater than the dose that can block tetrodotoxin-sensitive sodium channels, which are excessively activated by aconitine, tetrodotoxin toxicity becomes prominent, and the mortality rate increases because of the respiratory effects of tetrodotoxin. In terms of cardiotoxicity, mice receiving the aconitine-tetrodotoxin mixture showed minor and shorter periods of change on electrocardiography. This finding can be explained by the recent discovery of tetrodotoxin-sensitive sodium-channel cardiac isoforms (Na(v)1.1, 1.2, 1.3, 1.4 and 1.6).

Analysis of quazepam and its metabolites in human urine by gas chromatography-mass spectrometry: application to a forensic case.

A sensitive method for the simultaneous determination of quazepam and two of its metabolites, 2-oxoquazepam and 3-hydroxy-2-oxoquazepam, in human urine was developed using gas chromatography-mass spectrometry (GC/MS) with an Rtx-5MS capillary column. The quazepam and its metabolites were extracted from human urine using a simple solid-phase extraction Oasis(®) HLB cartridge column, and the 3-hydroxy-2-oxoquazepam was derivatised using BSTFA/1%TMCS and pyridine at 60 °C for 30 min. The mass spectrometric detection of the analytes was performed in the full scan mode, m/z 60-480, and selected ion monitoring (SIM) mode, m/z 386, for quazepam; m/z 342, for 2-oxoquazepam; m/z 429, for 3-hydroxy-2-oxoquazepam-TMS; and m/z 284, for alprazolam-d5 (internal standard), by electron ionization. The calibration curves of quazepam and its metabolites in urine showed good linearity in the concentration range of 2.5-500 ng/0.2 ml of urine. The average recoveries of quazepam and its metabolites from 0.2 ml of urine containing 500 ng and 50 ng of each drug were 71-83% and 88-90%, respectively. The limits of detection of quazepam, 2-oxoquazepam and 3-hydroxy-2-quazepam in urine by the selected ion monitoring mode were 0.096-0.37 ng/ml. This method would be applicable to other forensic biological materials containing low concentrations of quazepam and its metabolites.