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Z-drugs, benzodiazepines and ketamine are classes of psychotropic drugs prescribed for treating anxiety, sleep disorders and depression with known side effects including an elevated risk of addiction and substance misuse. These drugs have a strong potential for misuse, which has escalated over the years and was hypothesized here to have been exacerbated during the COVID-19 pandemic. Wastewater-based epidemiology (WBE) constitutes a fast, easy, and relatively inexpensive approach to epidemiological surveys for understanding the incidence and frequency of uses of these drugs. In this study, we analyzed wastewater (n = 376) from 50 cities across the United States and Mexico from July to October 2020 to estimate drug use rates during a pandemic event. Both time and flow proportional composite and grab samples of untreated municipal wastewater were analyzed using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry to determine loadings of alprazolam, clonazepam, diazepam, ketamine, lorazepam, nordiazepam, temazepam, zolpidem, and zaleplon in raw wastewater. Simultaneously, prescription data of the aforementioned drugs were extracted from the Medicaid database from 2019 to 2021. Results showed high detection frequencies of ketamine (90 %), lorazepam (87 %), clonazepam (76 %) and temazepam (73 %) across both Mexico and United States and comparatively lower detection frequencies for zaleplon (22 %), zolpidem (9 %), nordiazepam (<1 %), diazepam (<1 %), and alprazolam (<1 %) during the pandemic. Average mass consumption rates, estimated using WBE and reported in units of mg/day/1000 persons, ranged between 62 (temazepam) and 1100 (clonazepam) in the United States. Results obtained from the Medicaid database also showed a significant change (p < 0.05) in the prescription volume between the first quarter of 2019 (before the pandemic) and the first quarter of 2021 (pandemic event) for alprazolam, clonazepam and lorazepam. Study results include the first detections of zaleplon and zolpidem in wastewater from North America., Competing Interests: Declaration of competing interest R.U.H. and E.M.D. are cofounders of AquaVitas, an Arizona State University startup company providing commercial services in wastewater-based epidemiology. R.U.H. also is the founder of OneWaterOneHealth, a nonprofit project of the Arizona State University Foundation. B.J. Piper is part of an osteoarthritis research team supported by Pfizer and Eli Lilly., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pharmaceuticals as environmental contaminants have received a lot of interest over the past decade but, for several pharmaceuticals, relatively little is known about their occurrence in European surface waters. Benzodiazepines, a class of pharmaceuticals with anxiolytic properties, have received interest due to their behavioral modifying effect on exposed biota. In this study, our results show the presence of one or more benzodiazepine(s) in 86% of the analyzed surface water samples (n = 138) from 30 rivers, representing seven larger European catchments. Of the 13 benzodiazepines included in the study, we detected 9, which together showed median and mean concentrations (of the results above limit of quantification) of 5.4 and 9.6 ng L -1 , respectively. Four benzodiazepines (oxazepam, temazepam, clobazam, and bromazepam) were the most commonly detected. In particular, oxazepam had the highest frequency of detection (85%) and a maximum concentration of 61 ng L -1 . Temazepam and clobazam were found in 26% (maximum concentration of 39 ng L -1 ) and 14% (maximum concentration of 11 ng L -1 ) of the samples analyzed, respectively. Finally, bromazepam was found only in Germany and in 16 out of total 138 samples (12%), with a maximum concentration of 320 ng L -1 . This study clearly shows that benzodiazepines are common micro-contaminants of the largest European river systems at ng L -1 levels. Although these concentrations are more than a magnitude lower than those reported to have effective effects on exposed biota, environmental effects cannot be excluded considering the possibility of additive and sub-lethal effects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pentobarbital is a barbiturate, acting as a central nervous system depressant (CNS), being used for its anticonvulsant, sedative, hypnotic and anaesthetic properties. Barbiturates were replaced by benzodiazepines, leading to a decrease in poisoning cases with these compounds. However, pentobarbital is still used in many countries as an anaesthetic in veterinary medicine. Due to its properties, this compound is sought after by people who wish to commit suicide, acquiring it on the black market. The authors present an unusual fatal pentobarbital intoxication case, in a 37 years-old male salesperson, with no known connection with the veterinary field, being more difficult to obtain this compound. Toxicological results in cardiac blood revealed the presence of pentobarbital (111mg/L), ethanol (0.94g/L), diazepam (33ng/mL), nordiazepam (50ng/mL), oxazepam (3.3ng/mL), temazepam (5.3ng/mL), and metoclopramide. No illicit drugs were detected. Pentobarbital analysis in urine and gastric content was also positive, as well as its presence in the glass powder and in the bottle residue sent to the laboratory. In the present case, it was possible to conclude that the death was a suicide due to pentobarbital intoxication in association with other depressants of the CNS (benzodiazepines and ethanol). It is important to search pentobarbital in routine toxicological analyses, since it is one of the drugs most frequently mentioned by entities defending "painless death", advising the simultaneous use of metoclopramide for emesis avoidance., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Defining a method development methodology for achiral drug impurity profiling in SFC requires a number of steps. Initially, diverse stationary phases are characterized and a small number of orthogonal or dissimilar phases are selected for further method development. In this paper, we focus on a next step which is the investigation of the modifier composition on chromatographic selectivity. A solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH), propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in terms of elution order, retention and mixture resolution. However, varying only the modifier composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore the modifier composition is an optimization parameter, with the stationary phase being the factor determining most the selectivity of a given mixture in achiral SFC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.

Background: A simple and fast modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method is presented for the determination of diazepam and its three major metabolites, nordiazepam, temazepam and oxazepam (benzodiazepines) in fish samples by liquid chromatography-electrospray ionisation-tandem mass spectrometry., Results: Muscle tissues were extracted with acetonitrile, and then cleaned with primary secondary amino (PSA) adsorbents. The cleanup effect of PSA was compared with that of multi-walled carbon nanotubes (MWCNTs) in term of extraction efficiency. The better results were obtained when PSA was used. The chromatography separation was achieved within 5.0 min on a C18 column. The limit of detection was 0.5 µg kg(-1) and the limit of quantification was 2.5 µg kg(-1). Average recoveries of diazepam and its main metabolites were in the range of 88.5-110.1%, with a relative standard deviation lower than 10.0%., Conclusion: The proposed method for fish samples gives good recoveries, linearity, precision and accuracy., (© 2015 Society of Chemical Industry.)

Background: In recent years, the use of herbal weight-loss products sold over the Internet has rapidly increased; however, the safety of these products has not been well documented yet. More importantly, the declared ingredients in these products could be different than the marketed contents., Methods: Nine different herbal weight-loss products sold over the Internet were obtained. The ingredients of each product were analyzed in the Laboratory of Forensic Medicine and the Scientific and Technological Research Laboratory of Inonu University., Results: Although all studied weight-loss products were presented as pure herbal, three of them contain sibutramine, three contain caffeine, and three contain caffeine + temazepam. The amount of sibutramine in each capsule was found to be over 10 mg. We analyzed toxic and trace element levels of nine herbal products and found that these herbal products, even in low amounts, contain Pb, Al, Ni, and Ba., Conclusions: Our results indicate that herbal weight-loss products available without prescription and claimed to be purely herbal may contain pharmaceutical substances like sibutramine or temazepam in high doses. Moreover, they also may become contaminated with toxic metals. Since people commonly use these products unaware of its real constituents and without the suggestion or control of a physician, they might cause various health problems some of which might be harmful. Strict legal rules and control mechanisms must be established to minimize their possible harmful effects.

This paper reports an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method to quantitate 21 benzodiazepines, zolpidem and zopiclone in serum and plasma. After liquid-liquid extraction, an Acquity UPLC with a TQ Detector and BEH C18 column was used (Waters, Milford, MA). The injection-to-injection run time was 7.5 min. Forty-eight authentic serum and plasma patient specimens were analyzed and results compared to those obtained using a previously published method. Average r(2) values for linearity (1 to 1,000 ng/mL over five days) were all above 0.995, except α-hydroxytriazolam (0.993). Intra-day and inter-day relative standard deviation values were within ± 15% and the percent deviation from the expected concentrations were within ± 11%. Recovery ranged from 62 to 89%. Matrix effects ranged from -28% to +6%. The limits of detection were 1 ng/mL, except for lorazepam, nordiazepam, oxazepam and temazepam (5 ng/mL). Ion ratios were ± 15% for all analytes. For authentic patient specimens (n = 48, 76 positive results), there was excellent correlation between the UPLC-MS-MS results and the previous method. The best least-squares fit had an equation of y = 1.0708x + 1.6521, r(2) = 0.9822. This UPLC-MS-MS method is suitable for the quantification of benzodiazepines and hypnotics in serum and plasma, and offers fast, reliable and sensitive results.

Background: Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS) provides the ability to identify a range of benzodiazepines in accordance with European Union criteria and is an attractive method for the confirmation of benzodiazepines following immunoassay screening., Methods: An LC/MS/MS method to detect and quantitate the six most common benzodiazepines/metabolites (diazepam, nitrazepam, nordiazepam, oxazepam, temazepam and 7-aminonitrazepam) was developed together with a qualitative screening method for a further 11 benzodiazepines/metabolites. These methods were used for confirmation of 250 urine samples submitted for routine drug screening by immunoassay for benzodiazepines (100 samples positive for a benzodiazepine, assay cut-off >200 microsg/L)., Results: The lower limits of detection and quantitation were less than 2.5 and 5 microg/L for the six most common benzodiazepines. Recoveries ranged between 97% and 102% and calibration curves were linear to at least 4000 microg/L (r = 0.99). Intra and inter-assay imprecision were <10% (n = 10) and <20% (n = 15), respectively. Confirmation of benzodiazepines using LC/MS/MS was achieved for 89% of the immunoassay-positive urine samples. Of the immunoassay-negative urine samples, 31% of these demonstrated a benzodiazepine using LC/MS/MS., Conclusion: The validated LC/MS/MS method developed is effective for the confirmation of immunoassay screening results for benzodiazepines. The lower limit of detection and assay specificity offers a longer window of detection and more detailed clinical information compared with immunoassay screening.

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to "normal" rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n=134) and drug-free individuals (n=137), using the Cozart DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n=271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart DDS buffer and drug recovery was determined by Cozart enzyme immunoassays. The recovery studies showed that for amphetamine, Delta(9)THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.

A solid phase extraction (SPE) method has been developed and applied in conjunction with a previously reported liquid chromatography tandem mass spectrometry (LC-MS-MS) procedure for the determination of illicit drugs and abused pharmaceuticals in treated wastewater and surface water samples at the ng L(-1) level. A full method validation was also performed and determined levels of analytical sensitivity were found to lie in the 1-10 ng L(-1) range using river water as a test sample matrix and a sample size of 500 mL. The developed procedure was successfully applied for the determination of the chosen analytes in wastewater treatment plants in Dublin, Ireland and rapidly expanding commuter towns in the surrounding counties. Cocaine was detected in 70% of the collected samples in the range of 25-489 ng L(-1), its primary metabolite, benzoylecognine (BZE) was also detected in the range of 22-290 ng L(-1). Other substances detected included morphine, Tempazepam and the primary metabolite of methadone.

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.

A chiral amino acid-based monomeric and polymeric surfactant, sodium oleyl-L-leucylvalinate) (L-SOLV) and poly(sodium oleyl-L-leucylvalinate) (poly-L-SOLV) were synthesized and used for chiral separations in micellar electrokinetic chromatography (MEKC). Poly-L-SOLV was used successfully in the separation of various enantiomers of neutral, acidic, and basic analytes such as 1,1'-bi-2-napthol, 1,1'-binaphthyl-2,2'-diamine, benzoin, hydrobenzoin, benzoin methylether, warfarin, and coumachlor obtaining well-resolved peaks but with only partial separation of temazepam. In addition, the atropisomer 1,1'-binaphthyl-2, 2'-dihydrogen phosphate was chosen to study the applicability of the polymeric surfactant over a wide range of parameters such as concentration, temperature, voltage, and pH. The most striking characteristic of this new surfactant is its high hydrophobicity. It is favorable to interactions with hydrophobic chiral analytes, and thus may provide better chiral recognition for the compounds.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis., (Copyright 2002 Elsevier Science B.V.)

The temperature-dependent enantiomerization barriers of oxazepam, temazepam, and lorazepam have been determined between 0 and 30 degrees C by dynamic micellar electrokinetic chromatography (DMEKC) in an aqueous 20 mM borate/phosphate buffer system at pH 8 with 60 mM sodium cholate as chiral surfactant. Interconversion profiles featuring plateau formation and peak broadening were observed and simulated by the new program ChromWin based on the theoretical plate as well as on the stochastic model using the experimental data plateau height, hplateau, peak width at half-height, wh, total retention times, tR, and electroosmotic breakthrough time, t0. Peak form analysis yielded rate constants k and kinetic activation parameters, deltaG double dagger, deltaH double dagger, and deltaS double dagger, of the enantiomerization of oxazepam, temazepam, and lorazepam. At 25 degrees C, the enantiomerization barrier, deltaG double dagger, was determined to be approximately 90 kJ mol-1 and the half-lives, tau, were determined to be approximately 21 min. The new approach allows the fast and precise determination of enantiomerization barriers in a biogenic environment and it mimics physiological conditions, as no organic modifiers or abiotic chiral stationary phases (CSP) are employed.

We evaluated the homogeneity of drug concentrations in muscle in 14 cadavers, comprising 11 drug overdoses and three cases of chronic therapeutic drug use. Analyses were performed on samples from twelve named muscles and femoral venous blood. Standard analytical techniques and instrumentation were used throughout. There was marked within-case variability in drug concentrations with highest:lowest concentrations ranging up to 21.7. Overall highest concentrations were found in the diaphragm and mean diaphragm:blood ratios ranged from 1.1 (temazepam, two cases) and 1.2/1.3 (paracetamol, six cases) up to 6.5/13.5 (amitriptyline, three cases) and 5.3/21.3 (propoxyphene, four cases). Excluding the diaphragm, mean muscle:blood ratios ranged from 0.4 (prothiaden), 0.5 (temazepam), and 0.7 (paracetamol) up to 3.7 (temazepam), 4.3 (propoxyphene) and 5.7 (amitriptyline). We suggest that muscle is suitable for qualitative analysis but not for quantitative corroboration of a blood sample or as a quantitative alternative to blood.

Current methods for studying in vitro drug metabolism involve add-incubate-separate-measure approach. Separation of the desired analytes requires removal of protein which is typically accomplished by precipitation and centrifugation and extraction of the analytes into an organic phase. The analysis scheme then becomes more complex resulting in a decrease in precision and an increase in assay time. Microdialysis sampling circumvents these problems by allowing researchers to sample the reaction mixture periodically and obtain the complete metabolic profile. In the present study, microdialysis sampling was used to investigate Phase I metabolism of salicylic acid, diazepam and ibuprofen in rat liver microsomes. The major metabolites of these drugs were profiled by LC. Michaelis-Menten enzyme kinetic parameters, Km and Vmax were obtained for the formation of diazepam metabolites by both microdialysis and conventional microsomal incubations and were in good agreement with the values reported in the literature. This study shows that microdialysis has considerable promise as a sampling technique for in vitro drug metabolism studies. By making minor modifications to the instruments, microdialysis can be applied to other in vitro systems such as isolated hepatocytes to study the Phase II metabolism or tissue slices to study drug distribution.

The homogeneity of drug concentrations in skeletal muscle was assessed in eight fatal overdoses. Ten to 30 random samples were taken from leg muscle weighing 1,650 to 7,985 g. For cases involving paracetamol the mean muscle-to-blood ratio ranged from 0.1 to 1.1 (n = 4) for amitriptyline 1.1 to 3.6 (n = 3), and for dothiepin 0.8 to 2.1 (n = 2). The coefficient of variance was large for all drugs, ranging from 10.5 (carbamazepine) to 50 (thioridazine). Skeletal muscle is not homogeneous with respect to drug concentrations in fatal overdose cases. Of 16 instances of drug detection in blood 2 (nortriptyline and promethazine) were not detected in muscle. Muscle-to-blood drug ratios varied significantly among cases, possibly influenced by survival time after drug ingestion. Quantitative interpretations of muscle drug levels present significant difficulties. However, skeletal muscle can be used for qualitative corroboration of blood analyses and is a suitable specimen for drug detection where none other is available.

During the development of a micellar electrokinetic chromatographic screening method for 1,4-benzodiazepines, peak splitting and broadening were observed for some 3-hydroxy-1,4-benzodiazepines (oxazepam, lorazepam, temazepam and lormetazepam). This phenomenon occurred when the micellar phase consisted of bile salts and can be ascribed to the chiral nature of these surfactants. As the bile salts were applied in order to reduce the capacity factors to an appropriate level, enantiomer separation was not an objective and even disturbing. By increasing the analysis temperature, the chiral recognition of these compounds could be suppressed.

Calliphora vicina larvae were fed on drug-laden muscle from three suicides involving amitriptyline, temazepam and a combination of trazodone and trimipramine; triplicate daily harvestings were analysed. The limit of detection for all four drugs was 0.01 micrograms drug/g larvae. Mean drug concentrations (microgram/g) in the initial muscle were:amitriptyline, 2.68; temazepam, 4.04; trazodone, 21.56; and trimipramine, 19.58. Larval rearings for days 4-8 (15 larval samples per drug) had mean and ranges of drug concentrations (microgram/g) of 0.10 (r, 0.02-0.24) for amitriptyline; 0.52 (r, 0.26-0.78) for temazepam; 0.13 (r, 0.05-0.32) for trazodone; and 0.28 (r, 0.10-0.59) for trimipramine. After day 8 there was a precipitous fall in larval drug concentrations associated with pupariation. At day 11 ranges of drug concentrations (microgram/g) were: amitriptyline, < 0.01-0.01; temazepam, 0.01-0.08; trazodone, < 0.01-0.01; and trimipramine, 0.04-0.04. Day 16 pupae had corresponding ranges (microgram/g) of < 0.01, 0.01-0.01, < 0.01 and < 0.01-0.02. Transfer to drug-free food at day 5 led to similar falls in drug concentrations (microgram/g) from day 5 to day 6: 0.08-0.03 for amitriptyline, 0.61-0.09 for temazepam, 0.13-0.01 for trazodone, and 0.30-0.02 for trimipramine. The results show considerable variation in larval drug concentrations, both at the same developmental stage and at different stages of the life cycle, under conditions which closely reflect case situations. In practice, the precipitous decrease in drug concentrations in non-feeding larvae and at pupariation make it desirable to sample only larvae actively feeding on a corpse.

A highly selective and sensitive fluorimetric method was developed for the determination of four 1,4-benzodiazepine drugs containing a hydroxyl group at carbon 3, namely oxazepam, lorazepam, cinolazepam and temazepam. The method is highly specific because other benzodiazepinee lacking the hydroxyl group at C-3 do not react similarly and hence do not interfere. The proposed method involves reduction of the target compound using Zno/HCl at room temperature with the formation of a highly fluorescent derivative within 15 min. The different experimental parameters were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is applicable over the concentration range of 0.1-1.2 micrograms ml-1 for both temazepam and cinolazepam, and 0.2-2.5 and 1-8 micrograms ml-1 for oxazepam and lorazepam respectively. The recoveries of the title compounds from spiked urine ranged from 90.0 to 92.0% and for serum from 94.1 to 95.4% with a limit of detection (S/N = 2) of 4 ng ml-1 for all drugs. The mechanism of the fluorimetric reaction is discussed.

Temazepam undergoes a rearrangement reaction in strongly alkaline media to form a cyclic diamide, 7-chloro-1-methyl-5-phenyl-4,5-dihydro-2H-benzodiazepin-2,3(1H)-di one. Thermodynamic parameters (Eact, delta H++, delta S++, delta G++) involved in the rearrangement reaction, studied using either CH3CN-0.2 N NaOH in H2O (1:1, v/v) or CH3CN-0.2 N NaOD in D2O (1:1, v/v) as the solvent, were similar with an isotope effect (kH/kD) of 0.77 +/- 0.03. Kinetics of the rearrangement reaction were studied as a function of NaOH concentration, temperature and ionic strength. Results indicated that the rate-determining step did not involve proton exchange with solvent. Mass spectral analysis of the cyclic diamides derived by using either D2O or H2(18)O in the solvent mixtures suggested that the formation of the cyclic diamide involved a nucleophilic addition of a hydroxide ion at the C2 carbonyl carbon of temazepam.

Enantiomers of 3-O-methyloxazepam and 3-O-ethyloxazepam were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Temperature-dependent and acid-catalysed racemization of 3-O-methyloxazepam enantiomers in methanol and 3-O-ethyloxazepam enantiomers in ethanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of reaction product were determined by CSP-HPLC. Thermodynamic parameters in the formation of the activated complex (Eact, delta H++, delta S++ and delta G++) were consistent with those determined by a spectropolarimetric method. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer is proposed to be an intermediate and responsible for the acid-catalysed stereoselective homonucleophilic substitution and the resulting racemization.

The resolution of four racemic 3-hydroxy-1,4-benzodiazepin-2-ones, widely used in therapeutics, by means of a chiral stationary phase is described. The chiral selector used is (S)-N-(3,5-dinitrobenzoyl)phenylalanine. This chiral stationary phase showed both good enantioselectivity and efficiency for the compounds. Elution times were in all cases shorter than those previously reported for such compounds on different stationary phases. Racemic oxazepam was used to evaluate the loading capacity of the chiral stationary phase.

For studies on the excretion of drugs into milk a sensitive high-performance liquid chromatographic assay was developed to quantitate diazepam and nordazepam in the milk and plasma of humans and rabbits in the presence of their major metabolites, oxazepam and temazepam. Flurazepam was used as an internal standard. The assay involves extractions with diethyl ether and an additional acid clean-up step. Chromatographic separation was achieved by a LiChrospher 60 RP-select B (5 microns) column and KH2PO4- acetonitrile (69:31, v/v) adjusted to pH 2.80 as a mobile phase. The same extraction and chromatographic conditions were suited to both types of samples, milk and plasma. The limits of determination using ultraviolet detection at 241 nm was for diazepam 20 ng/ml and for nordazepam 15 ng/ml. The absolute recoveries of diazepam, nordazepam and flurazepam in human milk were 84, 86 and 92% and in human plasma 97, 89 and 94%, respectively. The within- and between-day accuracy and precision for diazepam and nordazepam in milk and plasma at all concentrations tested (20-1500 ng/ml) were better than 8%. The high fat content which occurs in rabbit milk presented no limitation for the extraction of lipophilic diazepam: the method was successfully used to monitor milk and plasma concentrations of diazepam and nordazepam in lactating New Zealand White rabbits during 26-h infusions of diazepam (1.4 mg/h).

The excretion of diazepam, N-desmethyldiazepam, temazepam and oxazepam in breast milk was studied during withdrawal of a 22-year-old patient from combined high dose diazepam and oxazepam therapy. Concentrations of these benzodiazepines in plasma from both the woman and her nursing infant (1 year old) were also documented. Diazepam, N-desmethyldiazepam, temazepam and oxazepam were found in the maternal plasma and milk with mean milk: plasma ratios of 0.2, 0.13, 0.14 and 0.10 respectively. It was calculated on a mg kg-1 basis that the infant received some 4.7% of the maternal dose. Diazepam could not be detected in the infant's plasma, but low levels of N-desmethyldiazepam (20 and 21 micrograms l-1), temazepam (7 micrograms l-1) and oxazepam (7.5 and 9.6 micrograms l-1) were present. The infant showed no overt physical or mental symptoms of benzodiazepine intoxication.

The advent of several new column materials for the resolution of chiral compounds in high-performance liquid chromatography has opened up new possibilities for the analysis of drug enantiomers both in the dosage form and in bioanalytical studies. The utility of simplex optimisation, modified simplex and response surface mapping are considered with reference to the antischistosomal drug, oxamniquine, separated on an alpha 1-acid glycoprotein column. The resolution of the enantiomers of three closely related benzodiazepines, temazepam, oxazepam and lorazepam, is attempted on three new column systems: cellulose triacetate, beta-cyclodextrin and the reversed-phase column porous graphitic carbon with beta-cyclodextrin as a mobile phase additive.

Two cases of lethal overdose by temazepam are presented. In both cases, temazepam was the only drug detected, and there was no involvement of alcohol. The drug concentrations in blood, urine, and liver were measured.

Acute intravenous (IV) injections of temazepam were examined for the ability to impair the performance of young (3-4-month-old), mature (12-15-month-old) and old (28-30-month-old) male Fischer 344 rats in the step-down task relative to vehicle-injected controls. The effect of temazepam on the passive-avoidance response could be characterized as a U-shaped function of age. The performance of the mature rat was not significantly impaired by an IV injection of temazepam between 18 and 320 micrograms/kg. Temazepam was more effective in impairing the performance of the young and old rat. The brain levels of temazepam after a single IV injection of 18 micrograms/kg in mature and senescent rats, and 32 micrograms/kg in young rats were measured over a 2-hour time period. The brain of the mature rat was exposed to less temazepam between 0 and 120 minutes than the brain of the old rat. Therefore, the increased sensitivity of the senescent rat relative to the mature rat may in part be due to changes in the pharmacokinetics of temazepam. However, the inability of temazepam (between 18 and 320 micrograms/kg) to impair the performance of mature rats in the passive-avoidance task suggests that pharmacodynamic changes may be involved in the decreased sensitivity of mature rats relative to young and senescent rats.

A reversed-phase, high-performance liquid chromatographic assay method is described for temazepam hard gelatin and soft gelatin capsule analysis. The method is simple, specific, accurate, fast, and stability indicating. A reversed-phase octylsilane (C8) column with a mobile phase composed of methanol:1% acetic acid and detection at 254 nm separated sulfanilamide (internal standard), temazepam, synthetic precursor, and possible degradation products. Detector responses showed linearity to temazepam concentrations over the range 0.075-0.60 mg/mL (r = 0.9999). Mean recovery of temazepam added to capsule excipients was 100.3%. Mean assay results for 15- and 30-mg hard gelatin capsules were 101.5 and 101.3%, respectively. Mean assay results for 10- and 20-mg soft elastic gelatin capsules were 101.1 and 101.5%, respectively.