Development of an Analytical Method for Detection of Anesthetics and Sedatives in Fish.

BACKGROUND: Anesthetics and sedatives are frequently used to prevent abrasions caused by stress and to facilitate fish management. However, drug residues may persist and cause changes in fish conditions and induce side effects. In addition, drugs that are not permitted for use in edible fish are sometimes potentially used in fish. The drugs can also be found in wastewater and are likely to be detected in fish. OBJECTIVE: The purpose of this study was to establish a quantitative analytical method for 10 anesthetic and sedative (azaperone, chlorpromazine, diazepam, estazolam, haloperidol, nitrazepam, nordiazepam, oxazepam, perphenazine, and temazepam) residues in fish sold in Korean markets. METHOD: Shrimp, flounder, and eel samples were selected as matrices. Acetonitrile (ACN) containing 0.1% formic acid was selected as an extraction solvent for shrimp and 100% ACN for flounder and eel. The QuEChERS method with C18 and primary secondary amine (PSA) was used as the extraction procedure, and the analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Limit of quantitation, recovery, accuracy, and precision were validated, and satisfactory results were obtained for the drugs. All results applied to the real samples were negative. CONCLUSIONS: An optimal validation method was studied. Since the results for all samples were negative, it is considered that additional studies are needed by increasing the number of drugs. HIGHLIGHTS: The most effective QuEChERS pretreatment method and conditions of LC-MS/MS for the analysis of anesthetics and sedatives in fish were established.

Development and Validation of an LC-MS-MS Method for the Detection of 40 Benzodiazepines and Three Z-Drugs in Blood and Urine by Solid-Phase Extraction.

An analytical method for the detection of 40 benzodiazepines, (±)-zopiclone, zaleplon and zolpidem in blood and urine by solid-phase extraction liquid chromatography-tandem mass spectrometry was developed and validated. Twenty-nine of 43 analytes were quantified in 0.5 mL whole blood for investigating postmortem, drug-facilitated sexual assault (DFSA) and driving under the influence of drugs cases (DUID). The four different dynamic ranges of the seven-point, linear, 1/x weighted calibration curves with lower limits of quantification of 2, 5, 10 and 20 µg/L across the analytes encompassed the majority of our casework encountered in postmortem, DFSA and DUID samples. Reference materials were available for all analytes except α-hydroxyflualprazolam, a hydroxylated metabolite of flualprazolam. The fragmentation of α-hydroxyflualprazolam was predicted from the fragmentation pattern of α-hydroxyalprazolam, and the appropriate transitions were added to the method to enable monitoring for this analyte. Urine samples were hydrolyzed at 55°C for 30 min with a genetically modified ß-glucuronidase enzyme, which resulted in >95% efficiency measured by oxazepam glucuronide. Extensive sample preparation included combining osmotic lysing and protein precipitation with methanol/acetonitrile mixture followed by freezing and centrifugation resulted in exceptionally high signal-to-noise ratios. Bias and between-and within-day imprecision for quality controls (QCs) were all within ±15%, except for clonazolam and etizolam that were within ±20%. All 29 of the 43 analytes tested for QC performance met quantitative reporting criteria within the dynamic ranges of the calibration curves, and 14 analytes, present only in the calibrator solution, were qualitatively reported. Twenty-five analytes met all quantitative reporting criteria including dilution integrity. The ability to analyze quantitative blood and qualitative urine samples in the same batch is one of the most useful elements of this procedure. This sensitive, specific and robust analytical method was routinely employed in the analysis of >300 samples in our laboratory over the last 6 months.

Establishment of pressurized liquid extraction followed by HPLC-MS/MS method for the screening of adrenergic drugs, steroids, sedatives, colorants and antioxidants in swine feed.

A facile and sensitive multi-residue detection approach of pressurized liquid extraction following high-performance liquid chromatography tandem mass spectrometry was established to detect the residues of adrenergic drugs, steroids, sedative, colorant and antioxidant in feed. The conditions employed for pressurized liquid extraction involved acetonitrile/ethyl acetate (1:1, v/v) as the extracting solvent, the temperature 80°C, two cycles and a static time of 10 min. The extraction was followed by a solid-phase extraction clean-up step. The separation of samples was done by C18 column with the mobile phase of 5 mM ammonium acetate solution and acetonitrile with 0.1% formic acid. The limits of quantification ranged from 0.03 to 1 µg/kg, limits of detection were in a range of 0.01-0.5 µg/kg, and average recoveries were 70.4-98.6%. The pressurized liquid extraction procedure was optimized and overall method was validated in terms of sensitivity, linearity, selectivity, matrix effect, accuracy, recovery and stability of the target drugs in the pressurized liquid extraction extracts solution. The screening method was proved to be fast, selective, accurate and sensitive for screening drugs.

Segmental Hair Analysis-Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment.

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, "false" positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results-especially concerning the time of drug intake-must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.

Neuropharmacological effects of essential oil from the leaves of Croton conduplicatus Kunth and possible mechanisms of action involved.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton conduplicatus Kunth (Euphorbiaceae) is a Brazilian aromatic medicinal plant, widely known as "quebra-faca". In folk medicine, its leaves and stem-barks are used as a natural analgesic for the treatment of headaches. AIM OF THE STUDY: In this study, we describe for the first time the neuropharmacological potential of the essential oil obtained from the leaves of Croton conduplicatus (EO) in experimental models of pain, anxiety and insomnia. The mechanisms of action involved in these activities were also investigated. MATERIAL AND METHODS: Different experimental models were used to evaluate the antinociceptive (acetic acid, formalin-induced nociception and hot plate tests), anxiolytic (elevated plus maze and hole board tests) and sedative (thiopental-induced sleeping time) effects of EO in mice. EO was evaluated in three different doses (25, 50 and 100 mg/kg, i.p.) and compared with positive and negative controls in all experimental protocols. When appropriate, animals were pretreated with pharmacological antagonists (naloxone, atropine and flumazenil) in order to evaluate the mechanisms of action involved. A docking study also was performed to identify possible targets involved. RESULTS: EO (25, 50 and 100 mg/kg, i.p.) demonstrated a significant antinociceptive activity in all experimental models. Pretreatment with naloxone or atropine reversed the antinociceptive response (p < 0.05), suggesting the involvement of opioid and muscarinic receptors, respectively. A docking study was performed with the major components identified in EO (1,8 cineole - 21.42%, spathulenol - 15.47%, p-cymene - 12.41% and caryophyllene oxide - 12.15%), demonstrating favorable interaction profile with different subtypes of muscarinic (M2, M3 and M4) and opioids (delta and mu) receptors. EO also showed anxiolytic (mainly at doses of 25 and 50 mg/kg, i.p.) and sedative (only at the dose of 100 mg/kg, i.p.) effects in mice. These pharmacological responses were reversed by flumazenil (p < 0.05), indicating possible involvement of GABAA receptors. CONCLUSION: Our findings support the traditional use of this plant as a natural analgesic and suggest that EO is a multi-target natural product, presenting not only antinociceptive effect but also anxiolytic and sedative activities depending on the dose used.

Determination of total and unbound propofol in patients during intensive care sedation by ultrafiltration and LC-MS/MS.

For the quantification of propofol total and unbound drug concentrations a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. To separate unbound propofol an ultrafiltration step before sample preparation was performed. Both the ultrafiltrate and plasma samples were extracted with solid-phase extraction and substituted with deuterated propofol as an internal standard. Separation was performed by gradient elution using UPLC-like system and analyzed by MS/MS consisting of an electrospray ionization source. To detect low and high concentration levels of propofol two calibration curves were identified and showed linearity within the range of 1-50ng/ml and 50-20000ng/ml. The lower limit of quantification was 1ng/ml. Intra- and interassay precision and accuracy did not exceed ±15%. The method was applied to a clinical study during intensive care treatment of patients after coronary artery bypass grafting.

Postmortem Propofol Levels: A Case of Residual Detection Long After Administration.

Propofol has gained notoriety in recent years because of its involvement in high-profile deaths and has increasingly become a drug of misuse and abuse particularly by health care personnel with easy access to it. In addition, propofol has also been used for more nefarious purposes such as murder and suicide. These, coupled with the drug's routine use for both major and minor medical procedures, provide ample opportunities for it to be implicated as a cause of death or contributing factor. In such instances, forensic investigators may be faced with the task of not only detecting the presence of propofol on postmortem toxicology screening, but also determining if it was indeed responsible for the decedent's demise. While propofol has a high volume of distribution, it is thought to equilibrate and be eliminated rapidly and not show significant tissue accumulation. However, this article presents a case illustrating that propofol can accumulate in the tissues and may be found up to a week after administration. This capacity to accumulate implies that postmortem detection does not necessarily confirm administration near the time of death, and further investigation needs to be undertaken to determine the timeline of events in order to rule out other factors, such as recent medical interventions, before attributing the cause of death to the presence of the drug.

Role of effective composition on antioxidant, anti-inflammatory, sedative-hypnotic capacities of 6 common edible Lilium varieties.

Nine Lilium samples (belong to 6 different cultivars with different maturity stage) were qualitatively and quantitatively analyzed of total phenolics (TP), total flavonoids (TF), total saponins (TS), total carbohydrates (TC, polysaccharides), and soluble proteins contents (SP), and the monomeric components were quantified utilizing high-performance liquid chromatography with photodiode array detector (HPLC-PAD) associated with liquid chromatography-mass spectrometry (HPLC-MS). Antioxidant activity (reducing power and DPPH radical scavenging activity), anti-inflammatory (xylene-induced mouse ear edema detumescent assay and carrageenan-induced mouse paw edema detumescent assay), and sedative-hypnotic capacities (sodium pentobarbital-induced sleep assay) were comparatively evaluated in mouse model. Additionally, correlation analysis and principal component analysis were carried out to detect clustering and elucidate relationships between components' concentrations and bioactivities to clarify the role of effective composition. Lilium bulbs in later maturity stage preliminary evidenced higher saponins content, and lower phenolic acids and flavonoids content. The result demonstrated that Lilium bulbs generally had distinct antioxidant, anti-inflammatory, and sedative-hypnotic capacities. Varieties statistically differed (P < 0.05) in chemical composition and bioactivities. Lilium varieties of Dongbei and Lanzhou presented potent sedative-hypnotic effect and anti-inflammatory activity. The antioxidant capacity was related to the phenolic acids and flavonoids contents, the anti-inflammatory and sedative-hypnotic capacities were related to the saponins content. This is first study presenting comprehensive description of common edible Lilium bulbs' chemical compositions, sedative-hypnotic, and anti-inflammatory capacities grown in China. It would informatively benefit the genetic selection and cultivated optimization of Lilium varieties to improve nutritional quality, and promote Lilium bulbs as a therapeutic functional food worldwide.

Detection of gaseous compounds by needle trap sampling and direct thermal-desorption photoionization mass spectrometry: concept and demonstrative application to breath gas analysis.

A fast detection method to analyze gaseous organic compounds in complex gas mixtures was developed, using a needle trap device (NTD) in conjunction with thermal-desorption photoionization time-of-flight mass spectrometry (TD-PI-TOFMS). The mass spectrometer was coupled via a deactivated fused silica capillary to an injector of a gas chromatograph. In the hot injector, the analytes collected on the NTD were thermally desorbed and directly transferred to the PI-TOFMS ion source. The molecules are softly ionized either by single photon ionization (SPI, 118 nm) or by resonance enhanced multiphoton ionization (REMPI, 266 nm), and the molecular ion signals are detected in the TOF mass analyzer. Analyte desorption and the subsequent PI-TOFMS detection step only lasts ten seconds. The specific selectivity of REMPI (i.e., aromatic compounds) and universal ionization characteristics render PI-MS as a promising detection system. As a first demonstrative application, the alveolar phase breath gas of healthy, nonsmoking subjects was sampled on NTDs. While smaller organic compounds such as acetone, acetaldehyde, isoprene, or cysteamine can be detected in the breath gas with SPI, REMPI depicts the aromatic substances phenol and indole at 266 nm. In the breath gas of a healthy, smoking male subject, several xenobiotic substances such as benzene, toluene, styrene, and ethylbenzene can be found as well. Furthermore, the NTD-REMPI-TOFMS setup was tested for breath gas taken from a mechanically ventilated pig under continuous intravenous propofol (2,6-diisopropylphenol, narcotic drug) infusion.

Quantitative analysis of quazepam and its metabolites in human blood, urine, and bile by liquid chromatography-tandem mass spectrometry.

Quazepam (QZP), which is a long-acting benzodiazepine-type hypnotic, and its 4 metabolites, 2-oxoquazepam, N-desalkyl-2-oxoquazepam (DOQ), 3-hydroxy-2-oxoquazepam (HOQ), and 3-hydroxy-N-desalkyl-2-oxoquazepam, in human blood, urine, and bile were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The analytes were extracted from blood by protein precipitation followed by solid phase extraction, and from urine and bile by liquid-liquid extraction and cleanup using a PSA solid phase extraction cartridge. This method was applied to a medico-legal autopsy case, in which the deceased had been prescribed QZP approximately 3 weeks before his death. In blood, the concentrations of free DOQ (160±7 ng/mL for heart blood and 181±12 ng/mL for femoral blood) were the highest of all the analytes and in agreement with the concentration at a steady state. This indicates that the deceased consecutively received QZP for at least several days until the concentrations reached approximately the same level as that in the steady state. An extremely high concentration of total HOQ (the sum of conjugated and free HOQ) in bile was also found (56,200±1900 ng/mL). This accumulation of HOQ in bile is probably due to enterohepatic circulation. This study demonstrates that the combination of the concentrations of QZP and its metabolites in biological matrices can provide more information about the amount and frequency of QZP administration.

Health worker exposure risk during inhalation sedation with sevoflurane using the (AnaConDa®) anaesthetic conserving device.

INTRODUCTION AND OBJECTIVE: Occupational exposure to sevoflurane should not exceed 2 ppm. During inhalation sedation with sevoflurane using the anaesthetic conserving device (AnaConDa(®)) in the post-anaesthesia care unit, waste gases can be reduced by gas extraction systems or scavenging devices such as CONTRAfluran™. However, the efficacy of these methods has not been clearly established. To determine the safest scenario for healthcare workers during inhalation sedation with sevoflurane in the post-surgical intensive care unit. MATERIALS AND METHODS: An experimental study on occupational exposure was conducted in a post-cardiothoracic care unit during March-August 2009. The measurements were performed in four post-cardiac surgery sedated adults in post-surgical intensive care unit and four nurses at the bedside, and at four points: scenario A, inhalation sedation without gas extraction system or contrafluran as a reference scenario; scenario B, applying a gas extraction system to the ventilator; scenario C, using contrafluran; and scenario 0, performing intravenous isolation sedation. Sevoflurane concentrations were measured in the nurses' breathing area during patient care, and at 1.5 and 8 m from the ventilator using diffusive passive monitor badges. RESULTS: All badges corresponding to the nurses' breathing area were below 2 ppm. Levels of sevoflurane detected using prevention systems were lower than that in the control situation. Only one determination over 2 ppm was found, corresponding to the monitor placed nearest the gas outlet of the ventilator in scenario A. Trace concentrations of sevoflurane were found in scenario 0 during intravenous sedation. CONCLUSIONS: Administration of sevoflurane through the AnaConDa(®) system during inhalation sedation in post-surgical intensive care units is safe for healthcare workers, but gas extraction systems or scavenging systems, such as CONTRAfluran™ should be used to reduce occupational exposure as much as possible.

Complex suicide with homemade nicotine patches.

Suicide by self-poisoning is rather common around the world. This paper presents an exceptional complex suicide in which nicotine was applied in the form of self-made patches soaked with an extraction from fine-cut tobacco. In addition, the 51-year-old suicide victim took a lethal dose of diphenhydramine. Toxicological analysis also revealed the presence of tetrazepam in subtherapeutic concentrations. The scene of death suggested an autoerotic accident at first, as the body was tied with tapes, cables and handcuffs. As a result of the entire investigations, the fatality had to be classified as a suicidal intoxication by nicotine and diphenhydramine.

Sedative and clinical effects of the pharmacopuncture with xylazine in dogs

To investigate the sedative and clinical effects of the pharmacopuncture with xylazine, compared to the conventional dose of a intramuscular injection in dogs. METHODS: Twelve dogs were randomly distributed in two groups of six animals and treated as follows: control group (X-IM): 1mg kg-1 of xylazine given intramuscularly (IM); pharmacopuncture group (X-Yintang): 0.1mg kg-1 of xylazine diluted to 0.5 mL of saline injected into the Yin Tang acupoint. Heart rate, cardiac rhythm (ECG), systolic arterial blood pressure (SABP), respiratory rate (RR), rectal temperature (RT), blood glucose concentration, degree of sedation and adverse effects were evaluated. RESULTS: Sedative effect was observed in both groups. The degree of sedation was greater in X-IM only at 15 min when compared with X-Yintang group. Cardiovascular established was observed in X-Yintang group, while marked reduction in the HR and increased incidence of ECG abnormalities were detected in X-IM. In both treatment groups, minimal changes were observed in relation to SABP, RR, RT and blood glucose. High incidence (66%) of vomiting was observed in X-IM, while this adverse effect was absent in X-Yintang. CONCLUSION: Pharmacopuncture with xylazine induced clinically relevant sedative effects in dogs, with the advantage of reduction of undesirable side effects associated with α2-agonists, including bradycardia, cardiac arrhythmias, and emesis.

Efficacy of eugenol and the methanolic extract of condalia buxifolia during the transport of the silver catfish Rhamdia quelen

This study evaluated extracts of Condalia buxifolia as anesthetics for the silver catfish Rhamdia quelen. The effectiveness of eugenol and of the methanolic extract (ME) of C. buxifolia during the transport of this species was also assessed. Fish of two different weights (1.50±0.02 g and 165.70±22.50 g) were transferred to aquaria containing water with the C. buxifolia ME or with fractions obtained from the ME, such as the n-hexane, dichloromethane, ethyl acetate, n-butane and aqueous fractions, at concentrations from 0-300 °L L-1. The C. buxifolia ME in the 0.5-120 °L L-1 range caused only light sedation, and the fractions did not have an effect on the fish. In the second experiment, another group of fish was transported for 12 h in 15 plastic bags. The fish were divided into five groups: control, 1 or 2.5 °L L-1 eugenol and 25 or 50 °L L-1 C. buxifolia ME. The non-ionized ammonia levels were lower at the end of transport in the groups with the compounds than in that with water alone. Moreover, both compounds decreased the Na+, Cl-, and K+ net effluxes; therefore, their addition to the water during transport is advisable because they reduce fish mortality and ion loss.

Este estudo investigou extratos de Condalia buxifolia como anestésico para jundiá Rhamdia quelen, e também a eficiência do eugenol e do extrato metanólico (EM) de C. buxifolia para utilização durante o transporte dessa espécie. Peixes de dois diferentes pesos (1,50±0,02 g e 165,70±22,50 g) foram transferidos para aquários contendo água com o EM de C. buxifolia ou frações obtidas a partir do EM (n-hexano, acetato de diclorometano, etil n- butano e aquoso, em concentrações na faixa de 0 - 300 °L L-1. O EM de C. buxifolia em concentrações na faixa de 0,5 - 120 °L L-1 causou somente uma sedação leve e as frações não tiveram efeito. No segundo experimento outro grupo de peixes foi transportado por 12 h em 15 sacos plásticos divididos em cinco tratamentos: controle, 1 ou 2,5 °L L-1 de eugenol e 25 ou 50 °L L-1 de EM de C. buxifolia. Os níveis de amônia não-ionizada foram menores nos tratamentos com ambos compostos em relação à água (controle). Além disso, ambos compostos diminuíram os efluxos líquidos de Na+, Cl- e K+ e, portanto, sua adição na água de transporte é aconselhável, pois reduzem a mortalidade e a perda de íons dos peixes.

Anesthesia and transport of fat snook centropomus parallelus with the essential oil of Nectandra megapotamica (Spreng) Mez

This study analyzed the chemical composition and anesthetic potential of essential oil (EO) of Nectandra megapotamica in fat snook (Centropomus parallelus). For the extraction of EO by hydrodistillation, leaves were separated in young (EO-Y) or old (EO-O), and the chemical composition of the EOs was determined by CG-MS. The anesthetic potential was assessed by the evaluation of induction and recovery time of anesthesia and stress response from anesthesia and transport. Three experiments were carried out: i) four different concentrations of each EO were tested to evaluate anesthesia induction and recovery time; ii) two concentrations of EO-O were tested for the evaluation of its effects on stress parameters (glucose, lactate, and Na+ and K+ plasma levels) caused by anesthesia; and iii) fish were transported in plastic bags, supplied with two concentrations of EO-O for the evaluation of water quality and mortality. All experiments were performed on fish acclimated to 0 and 33 ppt salinity. The main constituents of the Y and O-EOs were bicyclogermacrene (46.5/34.6%), α-pinene (26.8/26.2%), β-pinene (7.9/12.3%), and germacrene D (9.6/9.1%). Mild sedation was achieved at 30 °L L-1(1.3-3.2 min) and deep anesthesia at 150 °L L-1 (5.6-8.0 min) with both EOs. The recovery time ranged from 1-10 min. The EO-O was not able to avoid the stress of anesthesia evidenced by elevated glucose and lactate plasma levels observed in all groups. Plasma levels of Na+ and K+ were not significantly affected by treatments. During transport, the use of EO-O did not prevent deterioration in water quality and the post-transport mortality. In conclusion, the EO of N. megapotamica has anesthetic activity in fat snook, but it was not able to prevent the stress of anesthesia and transport.

Este estudo analisou a composição química e o potencial anestésico do óleo essencial (OE) de Nectandra megapotamica em robalos-peva (Centropomus parallelus). Para a extração do OE por hidrodestilação, as folhas foram separadas em jovens (OE-J) ou velhas (OE-V) e a composição química foi determinada por CG-EM. O potencial anestésico foi acessado através da avaliação do tempo de indução e recuperação da anestesia e resposta ao estresse do procedimento anestésico e transporte. Foram realizados três experimentos: em primeiro lugar, quatro concentrações diferentes de cada OE foram testadas para avaliar o tempo de indução à anestesia e de recuperação; em segundo lugar, duas concentrações do OE-V foram testadas para avaliar os efeitos sobre os parâmetros de estresse (níveis plasmáticos de glicose, lactato, Na+ e K+) causados pelo procedimento anestésico; em terceiro lugar, os peixes foram transportados em sacos plásticos com duas concentrações do OE-V para avaliação da qualidade da água e mortalidade. Todos os experimentos foram realizados em peixes aclimatados à salinidade zero e 33. Os constituintes majoritários do OE-J e OE-V foram: biciclogermacreno (46,5/34,6%), α-pineno (26,8/26,2%), β-pineno (7,9/12,3%) e germacreno D (9,6/9,1%). Sedação leve foi alcançada com 30 °L L-1(1,3-3,2 min) e anestesia profunda a partir de 150 °L L-1 (5,6-8,0 min) com ambos OEs. O tempo de recuperação variou entre 1-10 min.O OE-V não foi capaz de evitar o estresse do procedimento anestésico, evidenciado pelos elevados níveis plasmáticos de glicose e lactato observados em todos os grupos. Os níveis plasmáticos de Na+ e K+ não foram significativamente afetados pelos tratamentos. Durante o transporte, o OE-V não impediu a deterioração da qualidade da água e a mortalidade pós-transporte. Concluindo, o OE de Nectandra megapotamica apresenta atividade anestésica em robalos-peva, mas não foi capaz de evitar o estresse do procedimento anestésico e transporte.

Recent increase in detection of alprazolam in Victorian heroin-related deaths.

OBJECTIVES: To examine the rate of detection of alprazolam among cases of heroin-related death (HRD) in Victoria, including the relationship between alprazolam supply and HRDs. DESIGN AND SETTING: Population-based study of community alprazolam supply in Victoria and HRDs reported to the Victorian coroner from January 1990 to December 2010. MAIN OUTCOME MEASURES: Number of prescriptions for alprazolam supplied; defined daily dose (DDD) per 1000 population per 04 of alprazolam; number of cases of HRD in which alprazolam was detected through postmortem toxicological testing. RESULTS: Alprazolam supply increased by 1426%, from 0.42 DDD/1000/04 in 1990, to 6.41 in 2010. For every 1 unit increase in DDD/1000/04, the proportion of cases of HRD in which alprazolam was detected increased at an incidence rate ratio of 2.4 (95% CI, 2.1-2.8; P < 0.001). Alprazolam was detected among increasing proportions of HRDs, from 5.3% in 2005 to a peak of 35.3% in 2009. CONCLUSION: The increase in detection of alprazolam among cases of HRD, particularly since 2005, and the disproportionate increase in prescribing of the high-dose 2 mg formulation compared with other formulations suggest a need to examine alprazolam prescribing and to identify inappropriate prescribing and the circumstances of diversion from licit to illicit use.